title: A cDNA cassette system for the synthesis of recombinant procollagens. Variants of procollagen II lacking a D-period are secreted as triple-helical monomers.
abstract: Currently there is a lack of experimental systems for defining the functional domains of the fibrillar collagens. Here we describe an experimental strategy that employs the polymerase chain reaction (PCR) to create a series of cDNA cassettes coding for seven separate domains of procollagen II. The system was used to prepare novel recombinant procollagens II from which one of the four repetitive D-periods of the triple helix was deleted. Four constructs, each lacking a different D-period, were expressed in stably transfected mammalian cells (HT-1080). Truncated procollagens of the predicted size were recovered from the medium. All were triple-helical as assayed by circular dichroism. Therefore, deletion of a complete D-period containing 234 amino acids does not destabilize the triple helix of homotrimeric collagen II as much as some naturally occurring mutations in the heterotrimeric monomer of collagen I that delete shorter sequences or that convert obligate glycine residues to residues with bulkier side chains. Moreover, the results suggest that the strategy developed here can be used to map in detail the binding sites on fibrillar collagens for other components of the extracellular matrix and for the binding, spreading and signaling of cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: W V
authors.initials: WV
authors.lastname: Arnold
authors.forename: A L
authors.initials: AL
authors.lastname: Sieron
authors.forename: A
authors.initials: A
authors.lastname: Fertala
authors.forename: H P
authors.initials: HP
authors.lastname: Bächinger
authors.forename: D
authors.initials: D
authors.lastname: Mechling
authors.forename: D J
authors.initials: DJ
authors.lastname: Prockop
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D006801:Humans; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D011347:Procollagen; D011994:Recombinant Proteins