Tyrosine sulfation of proteins from the human hepatoma cell line HepG2.
[35S]Sulfate labeling of the human hepatoma cell line HepG2 showed it to contain many sulfated proteins of diverse molecular weight range. The isolation of tyrosine O-sulfate indicated the supernatant fraction to contain a 5- to 7-fold higher level than the cellular fraction at the end of a 24-hr incubation. The proteins in the supernatant fraction were immunoprecipitated and examined for sulfation. Of 15 proteins tested, 7 were found to be sulfated as indicated by [35S]sulfate incorporation into proteins separated by NaDodSO4/PAGE and detected by autoradiography. The 35S-labeled bands were excised from the dried gel and subjected to extensive Pronase hydrolysis and the hydrolysates were analyzed for tyrosine [35S]sulfate by a two-dimensional procedure combining high-voltage electrophoresis and thin-layer chromatography [Liu, M. C. & Lipmann, F. (1984) Proc. Natl. Acad. Sci. USA 81, 3695-3698]. Of the sulfated proteins, three--fibrinogen, alpha-fetoprotein, and fibronectin--were found to contain tyrosine O-sulfate. The simultaneous presence of carbohydrate-bound sulfate, however, could not be exactly determined, but the other four [35S]sulfate-containing proteins--alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin, and transferrin--did not reveal any tyrosine O-sulfate and might be sulfated on their carbohydrate moieties.
M C
MC
Liu
S
S
Yu
J
J
Sy
C M
CM
Redman
F
F
Lipmann
D006528:Carcinoma, Hepatocellular / Q000032:analysis*; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000032:analysis; D006801:Humans; D008113:Liver Neoplasms / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D000509:alpha-Fetoproteins / Q000032:analysis
D009363:Neoplasm Proteins; D000509:alpha-Fetoproteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D005340:Fibrinogen