A strategy for rapid sequencing of heparan sulfate and heparin saccharides

Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and, variab le patterns of sulfation, especially in heparan sulfate (HS). HS and the st ructurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is a ccumulating that these interactions depend on specific saccharide sequences , but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress, We describe how EIS and heparin sacc harides can be sequenced rapidly hv using an integrated strategy with chemi cal and enzymic steps. Attachment of a reducing-end fluorescent tag establi shes a reading frame. partial selective chemical cleavage at internal,N-sul foglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations o f exosulfatases and exoglycosidases permits the selective removal of specif ic sulfates and monosaccharides from their nonreducing ends. PAGE of the pr oducts yields a pattern of fluorescent bands from which the saccharide sequ ence can be read directly. Data are presented on sequencing of heparin tetr asaccharides and hexasaccharides of known structure; these data show the ac curacy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an BS decasacchari de of unknown structure. Application and further development of this sequen cing strategy. called integral glycan sequencing, will accelerate progress in defining the structure-activiy relationships of these complex GAGs and l ead to important insights into their biological functions.