Sulfation of tyrosine residues is a biosynthetic modification of physiologically important plasma proteins including the fourth component of complement, coagulation factors V and VIII, fibronectin, and alpha2- antiplasmin. In several of these examples, it is recognized that their sulfation has major effects on activity. We hypothesize that sites of sulfation often serve as recognition elements and points of contact in protein-protein interactions. The current proposal examines this hypothesis and extends our previous work on the biosynthesis and function of tyrosine sulfate residues in plasma proteins. Objectives are: 1) To define the roles of tyrosine sulfate residues in interactions of coagulation, fibrinolytic, and complement components, 2) To examine the stoichiometry and physiological variation of tyrosine sulfation, and 3) To identify recognition signals that direct the sulfation of specific sites. Contributions of sulfate groups to interactions of plasma proteins with the proteinases thrombin, plasmin, and the C1s subcomponent of complement will be studied by comparing activities of sulfated and nonsulfated forms of proteins and of synthetic peptides corresponding to sites of sulfation. Activities will be assessed with functional complement and coagulation assays and with purified enzymes. The affinities and thermodynamics of the binding of peptides to proteins will be measured by titration calorimetry. It is not known whether there is physiological or pathological variation in the sulfation of proteins. This issue will be studied by measuring the tyrosine sulfate content of several plasma proteins and of urine from different individuals. Specific proteins will be purified by immunoaffinity methods and their tyrosine sulfate content assessed by amino acid analysis of base hydrolysates or by HPLC analysis of peptides produced by proteolytic or chemical cleavage. A series of synthetic peptides will be studied as substrates of tyrosylprotein sulfotransferase to determine how substrate sites are identified, to provide optimal substrates for assay and affinity purification of the enzyme, and to serve as a basis for designing inhibitors. Long term goals are to understand the physiological significance and variation of the sulfation of tyrosine residues in proteins. In addition, this work will provide basic information about interactions between coagulation and complement components.
