Bulleid N J, Wilson R, Lees J F
School of Biological Sciences, University of Manchester, U.K.
Biochem J. 1996 Jul 1;317 ( Pt 1)(Pt 1):195-202. doi: 10.1042/bj3170195.
Procollagen assembly is initiated within the endoplasmic reticulum by three alpha-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen "mini-gene' which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.
前胶原组装在内质网中起始，由三条α链通过其C-前肽（C末端前肽）缔合而成。为了研究前胶原单体在合成时缔合的条件，我们利用半透性细胞重建了前胶原折叠、组装和修饰的初始阶段。通过翻译一个缺少部分三螺旋结构域的III型前胶原“微型基因”，我们证明这些细胞能有效地进行羟基化的、三螺旋的前胶原三聚体的组装，并能鉴定折叠途径中特定的二硫键连接的中间体。缺乏在C-前肽内形成链间二硫键能力的突变链，在这个系统中仍能组装。此外，对形成的三聚体分子的表征表明，在C-端肽（C末端端肽）内形成了链间二硫键。然而，当脯氨酰和赖氨酰残基的羟基化被抑制时，在C-端肽中没有形成链间二硫键，这表明羟基化是三螺旋结构域初始成核所必需的。缺乏在C-前肽或C-端肽内形成链间二硫键能力的突变链，仍能组装形成通过N-前肽（N末端前肽）内的链间二硫键连接的三聚体三螺旋分子。这些结果表明，在C-前肽或C-端肽内形成链间二硫键对于链缔合和三螺旋形成不是必需的。
