The role of the α3(VI) chain in collagen VI assembly -: Expression of an α3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an α3(VI)-deficient cell line
The role of the α3(VI) chain in collagen VI assembly -: Expression of an α3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an α3(VI)-deficient cell line
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DOI：
10.1074/jbc.273.13.7423
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发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Bateman, JR
中科院分区：
文献类型：
--
作者：
Lamandé, SR;Sigalas, E;Bateman, JR
VI型胶原是一种微纤维蛋白，存在于几乎所有结缔组织的细胞外基质中，三个遗传上不同的亚基，即α1(VI)、α2(VI)和α3(VI)链，在细胞内结合形成三螺旋单体，然后在分泌之前组装成二硫键二聚体和四聚体，尽管序列考虑表明由这三个链组成的VI型胶原单体是最稳定的异构体，但VI型胶原的确切链组成仍然存在争议，也有人提出了只包含Al1(VI)和Alpha2(VI)链的替代组合。为了直接解决这个问题并研究α3(VI)链在组装中的作用，我们研究了缺乏α3(VI)合成的人骨肉瘤细胞系(SAOS-2)在生物合成和体外基质形成方面的特征，Northern分析显示SAOS-2细胞中有丰富的α1(VI)和α2(VI)mRNAs，但在SAOS-2细胞中没有检测到明显的α3(VI)mRNA，然而，在培养30天时，检测到少量的α3(VI)mRNA，尽管表达水平仍然远低于α1(VI)和α2(VI)。直到培养第30天，SAOS-2培养液或细胞层标本中才检测到VI型胶原蛋白，这表明尽管有丰富的α1(VI)和Alpha2(VI)合成，但没有稳定的VI型胶原蛋白产生，不表达Alpha3(VI)。在没有α3(VI)的情况下产生的α1(VI)和α2(VI)链是非螺旋的，并且大部分保留在细胞内并被降解。稳定地将α3(VI)cDNA表达构建的缺乏10个N末端A型亚域中的4个的SAOS-2细胞直接证明了α3(VI)链在VI胶原组装中的关键作用，转染组的Al3(VI)N6-C5链与内源性的Al1(VI)和Al2(VI)相关并形成了VI型胶原二聚体和四聚体，这些二聚体和四聚体被分泌和沉积在细胞外基质中，形成广泛的网络，这些数据表明，α3(VI)是形成稳定的胶原蛋白VI分子所必需的，N10-N7亚域不是分子组装所必需的。
Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues, Three genetically distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion, Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha 1(VI) and alpha 2(VI) chains have also been proposed, To address this question directly and study the role of the alpha 3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha 3(VI) production, Northern analysis showed an abundance of alpha 1(VI) and alpha 2(VI) mRNAs, but no detectable alpha 3(VI) mRNA was apparent in SaOS-2 cells, By day 30 of culture, however, small amounts of alpha 3(VI) mRNA were detected, although the level of expression was still much less than alpha 1(VI) and alpha 2(VI), Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha 1(VI) and alpha 2(VI), no stable collagen VI protein was produced without expression of alpha 3(VI). The alpha 1(VI) and alpha 2(VI) chains produced in the absence of alpha 3(VI) were non-helical and were largely retained intracellularly and degraded, The critical role of the alpha 3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha 3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains, The transfected alpha 3(VI) N6-C5 chains associated with endogenous alpha 1(VI) and alpha 2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix, These data demonstrated that alpha 3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.