ANALYSIS OF PROTEIN TYROSINE SULFATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sulfation is a widespread post-translational modification of tyrosines found in all animal cells containing a Golgi system. Our recent analysis of tyrosine sulfation sites showed that almost half contained the tyrosine trafficking motif, Y-x-x-Hydrophobic, (x being any amino acid). Since proteins containing either tyrosine-based site are processed in the trans-Golgi, what directs them to the correct destination? We have contrasted trafficking motifs with tyrosine sulfation sites, both containing the Y-x-x-hydrophobic pattern, to determine sequence features that distinguish these different tyrosine-based sites. Several log-odds position specific scoring matrices (PSSM) were created to distinguish sulfation sites from trafficking motifs. Sulfation sites and trafficking motifs were contrasted with each other, as well as against the Swiss-Prot metazoan database. Three of thirteen trafficking motifs scored in the range of known tyrosine sulfation sites. Evaluation of the PSSMs showed that the pattern of high-scoring amino acids differs for trafficking motifs and sulfation sites. In trafficking motifs, arg and gln are preferred at position y+1 and gly at y-1, whereas in sulfation sites small residues (asp, gly, pro) are preferred at y+1, and asp at y-1. Leu is preferred at y+3 in trafficking motifs, while met and trp are preferred in sulfation sites. These results indicate that although significant differences exist between tyrosine sulfation sites and trafficking motifs, there is some overlap between these two tyrosine-based sites. Further experiments should determine whether some trafficking motifs are sulfated in vivo, and whether sulfation affects a proteinsultimate destination. Additional factors such as additional motifs may be necessary to direct proteins to their correct destinations. In fact, approximately 1% of the tyrosines are sulfated in tissue in the rat. Despite this observation, only 24 unique protein sequences containing tyrosine sulfate have been described. The aim of this project is to analyze the amino acid sequences surrounding know tyrosine sulfation sites to determine the characteristics of this site and, therefore, to identify more candidates of sulfation. The specific goals are: 1. to identify the characteristics of sulfated tyrosines which distinguish them from non-sulfated tyrosines. These characteristics include the number and from non-sulfated tyrosines. These characteristics include the possible distribution of amino acids, as well as the possible secondary structural secondary structural features in the vicinity of the tyrosine. 2. to identify features in the vicinity of the tyrosine. 2. to identify proteins with probable proteins with probable tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in the vicinity of the the vicinity of the sulfation site. Identification of new protein sulfation sites may be essential for the understanding of the function of these proteins sulfation site. Identification of new protein sulfation sites may be essential and will lead to a better understanding of the function of tyrosine sulfation. for the understanding of the function of these proteins and will lead to a better understanding of the function of tyrosine sulfation. In fact, approximately 1% of the tyrosines are sulfated in tissue in the rat. Despite this observation, only 24 unique protein sequences containing tyrosine sulfate have been described. The aim of this project is to analyze the amino acid sequences surrounding know tyrosine sulfation sites to determine the characteristics of this site and, therefore, to identify more candidates of sulfation. The specific goals are: 1. to identify the characteristics of sulfated tyrosines which distinguish them from non-sulfated tyrosines. These characteristics include the number and from non-sulfated tyrosines. These characteristics include the possible distribution of amino acids, as well as the possible secondary structural secondary structural features in the vicinity of the tyrosine. 2. to identify features in the vicinity of the tyrosine. 2. to identify proteins with probable proteins with probable tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in the vicinity of the the vicinity of the sulfation site. Identification of new protein sulfation sites may be essential for the understanding of the function of these proteins sulfation site. Identification of new protein sulfation sites may be essential and will lead to a better understanding of the function of tyrosine sulfation. for the understanding of the function of these proteins and will lead to a better understanding of the function of tyrosine sulfation. In fact, approximately 1% of the tyrosines are sulfated in tissue in the rat. Despite this observation, only 24 unique protein sequences containing tyrosine sulfate have been described. The aim of this project is to analyze the amino acid sequences surrounding know tyrosine sulfation sites to determine the characteristics of this site and, therefore, to identify more candidates of sulfation. The specific goals are: 1. to identify the characteristics of sulfated tyrosines which distinguish them from non-sulfated tyrosines. These characteristics include the number and from non-sulfated tyrosines. These characteristics include the possible distribution of amino acids, as well as the possible secondary structural secondary structural features in the vicinity of the tyrosine. 2. to identify features in the vicinity of the tyrosine. 2. to identify proteins with probable proteins with probable tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in tyrosine sulfation sites from the Swiss Protein data base. 3. to determine sequence similarities among the set of known proteins in the vicinity of the the vicinity of the sulfation site. Identification of new protein sulfation sites may be essential for the understanding of the function of these proteins sulfation site. Identification of new protein sulfation sites may be essential and will lead to a better understanding of the function of tyrosine sulfation. for the understanding of the function of these proteins and will lead to a better understanding of the function of tyrosine sulfation. Publications - Chan, Steve, S., Nicholas, Hugh B., Jr., and Rosenquist, Grace L.: "Re-evaluation of the Determinants of Tyrosine Sulfation": (In manuscript) Achievements - 1. Statistical analysis of the amino acids surrounding the target tyrosine using a specific scoring matrix (PSSM) showed that the consensus sequence does not contain the information to predict tyrosine sulfation. Previously, tyrosine sulfation had been thought to involve recognition of speciic features of a consensus sequence by the sulfating enzyme, tyrosyl-protein sulfotransferase (TPST). 2. Accurate prediction of tyrosine sulfation requires consideration of all residues within a window of 5 amino acids on either side of the tyrosine. 3. We conclude that highly specific recognition features are not required for substrate recognition by TPST. TPST may instead broadly recognize and act on any tyrosine residue sufficiently exposed to the solvent. Publications - Chan, Steve, S., Nicholas, Hugh B., Jr., and Rosenquist, Grace L.: "Re-evaluation of the Determinants of Tyrosine Sulfation": (In manuscript) Achievements - 1. Statistical analysis of the amino acids surrounding the target tyrosine using a specific scoring matrix (PSSM) showed that the consensus sequence does not contain the information to predict tyrosine sulfation. Previously, tyrosine sulfation had been thought to involve recognition of speciic features of a consensus sequence by the sulfating enzyme, tyrosyl-protein sulfotransferase (TPST). 2. Accurate prediction of tyrosine sulfation requires consideration of all residues within a window of 5 amino acids on either side of the tyrosine. 3. We conclude that highly specific recognition features are not required for substrate recognition by TPST. TPST may instead broadly recognize and act on any tyrosine residue sufficiently exposed to the solvent. Publications - Chan, Steve, S., Nicholas, Hugh B., Jr., and Rosenquist, Grace L.: "Re-evaluation of the Determinants of Tyrosine Sulfation": (In manuscript) Achievements - 1. Statistical analysis of the amino acids surrounding the target tyrosine using a specific scoring matrix (PSSM) showed that the consensus sequence does not contain the information to predict tyrosine sulfation. Previously, tyrosine sulfation had been thought to involve recognition of speciic features of a consensus sequence by the sulfating enzyme, tyrosyl-protein sulfotransferase (TPST). 2. Accurate prediction of tyrosine sulfation requires consideration of all residues within a window of 5 amino acids on either side of the tyrosine. 3. We conclude that highly specific recognition features are not required for substrate recognition by TPST. TPST may instead broadly recognize and act on any tyrosine residue sufficiently exposed to the solvent. Publications - Nicholas, Jr., H.B., Chan, S.S., Rosenquist, G.L.: "Reevaluation of the Determinants of Tyrosine Sulfation": Endocrine: 11:285-292: 1999.