Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate.
Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion-exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post-translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres.
D002478:Cells, Cultured; D011232:Chemical Precipitation; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry; D003012:Microbial Collagenase / Q000494:pharmacology; D010434:Pepsin A; D011347:Procollagen / Q000378:metabolism*; D011863:Radioimmunoassay; D013431:Sulfates / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000096:biosynthesis
D011347:Procollagen; D013431:Sulfates; C005662:tyrosine O-sulfate; D014443:Tyrosine; D010434:Pepsin A; D003012:Microbial Collagenase