Tyrosine sulfation site is located in the C-terminal fibrin-binding domain in secreted fibronectin from rat embryo fibroblasts, line 3Y1.
Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D006868:Hydrolysis; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D005353:Fibronectins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D014357:Trypsin