title: Identification of the site of sulfation of the fourth component of human complement.
abstract: The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002396:Catechols / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003181:Complement C4 / Q000032:analysis*; D003864:Depression, Chemical; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D010455:Peptides / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D002396:Catechols; D003181:Complement C4; D010455:Peptides; C005662:tyrosine O-sulfate; D014443:Tyrosine; C034221:catechol