In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites.
To gain insight into the structural requirements for tyrosine sulfation in vivo, we have constructed and expressed an artificial gene encoding a polypeptide substrate for tyrosylprotein sulfotransferase. This gene codes for a protein, referred to as sulfophilin, which consists of a 12-times repeated heptapeptide unit corresponding to the identified tyrosine sulfation site of chromogranin B (secretogranin I), Glu-Glu-Pro-Glu-Tyr-Gly-Glu. The gene was fused to the signal sequence of secretogranin II to direct the sulfophilin protein to the secretory pathway. Stable expression of the artificial gene in NIH 3T3 cells resulted in the secretion of sulfated sulfophilin. Analysis of the stoichiometry of sulfation revealed that each of the 12 tyrosyl residues in sulfophilin was sulfated. Remarkably, up to 50% of the total protein-bound tyrosine sulfate secreted by the cells was contained in sulfophilin. The results indicate that the structural information contained in the heptapeptide motif is sufficient for stoichiometric tyrosine sulfation to occur in the living cell.
Niehrs
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Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Huttner
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WB
Rüther
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005813:Genes, Synthetic; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D009838:Oligodeoxyribonucleotides; D011506:Proteins; C085675:sulfophilin; C075838:presulfophilin; C005662:tyrosine O-sulfate; D014443:Tyrosine; D004247:DNA