A Cluster Sequencing Strategy To Determine the Consensus Affinity Domains in Heparin for Its Binding to Specific Proteins.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.