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.