Detection of procollagen biosynthesis using peptide-specific antibodies.
Peptides corresponding to selected sequences of the alpha 1 chain of the COOH propeptide of type I and type III human procollagen were synthesized and used as antigens to develop polyclonal and monoclonal antibodies. The antibodies were shown to be epitope specific using a peptide-based solid phase enzyme-linked immunoadsorbent assay. The antibodies were specific for the appropriate procollagens and the COOH propeptides isolated from serum-free culture supernatants of human skin fibroblasts. The rabbit antisera directed to the type I synthetic peptide bound the intact procollagen molecule and both the procollagen alpha 1(I) and alpha 2(I) chains after the reduction of the disulfide bonds. In addition, the antisera bound intact type I COOH propeptide, generated by bacterial collagenase treatment of procollagen, and the individual chains of the propeptide after reduction. In contrast, a monoclonal antibody to the type I peptide was able to bind only to the reduced form of the COOH propeptide. Both rabbit polyclonal and murine monoclonal antibodies directed to the type III synthetic peptide bound the intact and the individual chains of type III procollagen as well as the intact and reduced forms of the type III COOH propeptide. The antibodies have been used to detect procollagen synthesis in two human osteosarcoma cell lines and the differential expression of procollagen in the culture medium of rat lung fibroblasts grown in the presence or absence of glucocorticoids.
Mintz
K P
KP
Department of Biochemistry, University of Vermont, Burlington 05405.
Mann
K G
KG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002478:Cells, Cultured; D000939:Epitopes; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011347:Procollagen / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005938:Glucocorticoids; D011347:Procollagen