Abstract
Procollagen chains assemble in a type-specific manner forming either homo- or heterotrimers. The molecular mechanisms underlying procollagen chain selectivity are unknown, although it is thought that the C-propeptide (COOH-terminal propeptide) is responsible for directing chain recognition and assembly. To define the processes involved in chain selection we reconstituted the initial stages of procollagen folding and assembly in a cell-free system. Using human pro-alpha 1(III) and pro-alpha 2(I) chains as prototypes of chains that are either capable or incapable of forming homotrimeric molecules, respectively, we constructed two minigenes (p alpha 1(III) delta 1 and p alpha 2(I) delta 1) that lacked most of the triple helical domains. The minigenes were transcribed in vitro and translated in a rabbit reticulocyte lysate supplemented with microsomal membranes under conditions that favored disulfide bond formation. Both pro-alpha 1(III) delta 1 and pro-alpha 2(I) delta 1 chains formed intrachain disulfide bonds within the C-propeptide. However, only pro-alpha 1(III) delta 1 chains were able to self-associate forming homotrimers stabilized by interchain disulfide bonds. The C-propeptide of the pro-alpha 1(III) chain contains 8 cysteine residues (Cys-1-8). We used a site-directed mutagenesis to investigate the role of specific cysteine residues in trimer formation and found that substitution of serine for Cys-1, Cys-2, Cys-3, and Cys-4 prevented interchain disulfide bonding and trimerization. Furthermore, mutations in Cys-1 and Cys-4 also prevented intrachain disulfide bond formation within the C-propeptide. The C-propeptide of the pro-alpha 2(I) chain contains only 7 cysteine residues, lacking cysteine at position 2. Substitution of the existing Ser residue with Cys did not produce a homotrimeric phenotype, indicating that additional recognition signals are required to determine chain selection.
Highlights
The Roleof Cysteine Residuesin theFolding and Association of the COOH-terminal Propeptide of Types I and I11 Procollagen*
We have shown that pro-al(II1)monomers are able t o self-associate, forming disulfide-bonded trimers, whereas pro-a2(1) chains are unable t o form homotrimers.The C-propeptide of fibrillar collagens containsseveral highly conserved cysteine residues that participate in the formation of a number of intra- and interchaidnisulfide bonds
Tal plasmid was religated to create pal(II1)Al.Plasmid prr2U)Al was constructed from two partial cDNA clones, pHfll31 and pHp2010(13), The abbreviationsused are: C-propeptide and N-propeptide, COOHterminal propeptide and NH,terminal propeptide, respectively; D m, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair; C-proteinase, COOH-terminal proteinase
Summary
Construction of Recombinant Plasmids -A full-length type I11 procollagen cDNA was constructed from two partial cDNAs, pS413and S31 [12]. 34517).The costs of publication of this article were defrayed in part by the payment of page charges. Tal plasmid was religated to create pal(II1)Al.Plasmid prr2U)Al was constructed from two partial cDNA clones, pHfll and pHp2010(13), The abbreviationsused are: C-propeptide and N-propeptide, COOHterminal propeptide and NH,-terminal propeptide, respectively; D m , dithiothreitol; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair; C-proteinase, COOH-terminal proteinase.
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