Secretion and extracellular processing of procollagen by cultured human fibroblasts.
Cultures of human diploid fibroblasts were labeled with radioactive proline and glycine, and the precursor of collagen (procollagen) in cells and medium was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A covalently assembled molecule with the composition (pro alpha1)(2).pro alpha2 (approximate molecular weight, 360,000) appeared intracellularly soon after synthesis of the constituent chains, and could be detected in the medium after 60 min of labeling. The molecule was stabilized by disulfide bonds between cysteine residues in the amino-terminal procollagen peptide sequences of the three chains. Collagenase digested the molecule to peptides of 30,000 molecular weight or less. Limited digestion with pepsin excised nonhelical procollagen peptides, yielding native, triple-helical tropocollagen. Pulse-chase experiments indicated that a peptidase in the medium sequentially excised the nonhelical peptides from the molecule, generating tropocollagen molecules that aggregated as fibers in the cell layer. The excised, nonhelical procollagen peptides contained little or no proline or glycine. Intramolecular bonds of the lysyl aldehyde type were not detected in the secreted molecule, as reduction of the medium always resulted in quantitative recovery of free pro alpha chains in dodecyl sulfate-urea. Lysyl-derived, covalent bonds appeared to form between tropocollagen molecules aggregating in the cell layer. We suggest the term "pro-tropocollagen" for the assembled, secreted precursor of collagen.
D002247:Carbon Isotopes; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D003012:Microbial Collagenase / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D014316:Tritium
D002247:Carbon Isotopes; D011498:Protein Precursors; D014316:Tritium; D003094:Collagen; D011392:Proline; D010434:Pepsin A; D003012:Microbial Collagenase; D005998:Glycine