title: Recombinant expression and structural and binding properties of alpha 1(VI) and alpha 2(VI) chains of human collagen type VI.
abstract: Full-length alpha 1(VI) and alpha 2(VI) cDNAs in an eukaryotic expression vector were used to obtain stably transfected human kidney cell clones and to purify these collagen-VI chains in substantial quantities from the culture medium. Both chains appeared mainly as monomers together with some dimers that were disulfide linked through their C-terminal globular domains. Despite sufficient hydroxylation of proline and lysine residues, the chains did not form a triple-helix, as shown by electronmicroscopy, CD spectra and pepsin sensitivity. Digestion of the chains with bacterial collagenase released the N-terminal and C-terminal globular domains, which were identified by their size and partial sequences. They showed a substantial content of alpha-helical conformation and a distinct globular structure after rotary shadowing. Antibodies could be raised that distinguished between the two chains and reacted with the globular domains. The alpha 2(VI) but not the alpha 1(VI) chain showed binding to a heparan sulfate proteoglycan (perlecan), fibronectin and pepsin-solubilized collagen VI. Purified globular domains did not bind these ligands indicating the localization of binding sites within the triple-helical domain. Both chains showed a distinct affinity for heparin but failed to bind to various collagen types.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Tillet
authors.forename: H
authors.initials: H
authors.lastname: Wiedemann
authors.forename: R
authors.initials: R
authors.lastname: Golbik
authors.forename: T C
authors.initials: TC
authors.lastname: Pan
authors.forename: R Z
authors.initials: RZ
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Mann
authors.forename: M L
authors.initials: ML
authors.lastname: Chu
authors.forename: R
authors.initials: R
authors.lastname: Timpl
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D016326:Extracellular Matrix Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen