Lees J F, Bulleid N J
University of Manchester, School of Biological Sciences, United Kingdom.
J Biol Chem. 1994 Sep 30;269(39):24354-60.
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.
前胶原链以特定类型的方式组装，形成同三聚体或异三聚体。尽管人们认为C-前肽（羧基末端前肽）负责指导链的识别和组装，但前胶原链选择性背后的分子机制尚不清楚。为了确定链选择所涉及的过程，我们在无细胞系统中重建了前胶原折叠和组装的初始阶段。分别使用人原α1(III)链和原α2(I)链作为能够或不能形成同三聚体分子的链的原型，我们构建了两个缺少大部分三螺旋结构域的小基因（pα1(III)δ1和pα2(I)δ1）。小基因在体外转录，并在添加微粒体膜的兔网织红细胞裂解物中在有利于二硫键形成的条件下进行翻译。原α1(III)δ1链和原α2(I)δ1链都在C-前肽内形成链内二硫键。然而，只有原α1(III)δ1链能够自我缔合形成由链间二硫键稳定的同三聚体。原α1(III)链的C-前肽含有8个半胱氨酸残基（Cys-1-8）。我们使用定点诱变来研究特定半胱氨酸残基在三聚体形成中的作用，发现用丝氨酸取代Cys-1、Cys-2、Cys-3和Cys-4可防止链间二硫键结合和三聚化。此外，Cys-1和Cys-4中的突变也阻止了C-前肽内链内二硫键的形成。原α2(I)链的C-前肽仅含有7个半胱氨酸残基，在第2位缺少半胱氨酸。用半胱氨酸取代现有的丝氨酸残基并未产生同三聚体表型，表明需要额外的识别信号来确定链的选择。
