Homogeneous Sulfopeptides and Sulfoproteins: Synthetic Approaches and Applications to Characterize the Roles of Tyrosine Sulfation on Biochemical Function

Post-translational modification of proteins plays critical roles in regulating structure, stability, localization, and function. Sulfation of the phenolic side chain of tyrosine residues to form sulfotyrosine (sTyr) is a widespread modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection. Tyrosine sulfation commonly occurs in sequences containing clusters of tyrosine residues and is incomplete at each site, resulting in heterogeneous mixtures of sulfoforms. Purification of individual sulfoforms is typically impractical. Therefore, the most promising approach to elucidate the influence of sulfation at each site is to prepare homogeneously sulfated proteins (or peptides) synthetically. This Account describes our recent progress in both development of such synthetic approaches and application of the resulting sulfopeptides and sulfoproteins to characterize the functional consequences of tyrosine sulfation. Initial synthetic studies used a cassette-based solid-phase peptide synthesis (SPPS) approach in which the side chain sulfate ester was protected to enable it to withstand Fmoc-based SPPS conditions. Subsequently, to address the need for efficient access to multiple sulfoforms of the same peptide, we developed a divergent solid-phase synthetic approach utilizing orthogonally side chain protected tyrosine residues. Using this methodology, we have carried out orthogonal deprotection and sulfation of up to three tyrosine residues within a given sequence, allowing access to all eight sulfoforms of a given target from a single solid-phase synthesis. With homogeneously sulfated peptides in hand, we have been able to probe the influence of tyrosine sulfation on biochemical function. Several of these studies focused on sulfated fragments of chemokine receptors, key mediators of leukocyte trafficking and inflammation. For the receptor CCR3, we showed that tyrosine sulfation enhances affinity and selectivity for binding to chemokine ligands, and we determined the structural basis of these affinity enhancements by NMR spectroscopy. Using a library of CCR5 sulfopeptides, we demonstrated the critical importance of sulfation at one specific site for supporting HIV-1 infection. Demonstrating the feasibility of producing homogeneously tyrosine-sulfated proteins, in addition to smaller peptides, we have used SPPS and native chemical ligation methods to synthesize the leech-derived antithrombotic protein hirudin P6, containing both tyrosine sulfation and glycosylation. Sulfation greatly enhanced inhibitory activity against thrombin, whereas addition of glycans to the sulfated protein decreased inhibition, indicating functional interplay between different post-translational modifications. In addition, the success of the ligation approach suggests that larger sulfoproteins could potentially be obtained by ligation of synthetic sulfopeptides to expressed proteins, using intein-based technology.