An α2(I) glycine to aspartate substitution is responsible for the presence of a kink in type I collagen in a lethal case of Osteogenesis Imperfecta
An α2(I) glycine to aspartate substitution is responsible for the presence of a kink in type I collagen in a lethal case of Osteogenesis Imperfecta
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DOI：
10.1016/s0945-053x(98)90109-3
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发表时间：
1998-12-01
期刊：
影响因子：
6.9
通讯作者：
Marini, JC
中科院分区：
文献类型：
--
作者：
Forlino, A;Keene, DR;Marini, JC
通过对培养的皮肤成纤维细胞合成的I型胶原进行生物化学和分子分析，以表征受致命性成骨不全影响的患者的缺陷。前胶原和胶原的SDS-Urea-PAGE显示宽的α 1（I）带、正常的α 2（I）带和在α 1和α 2之间等距迁移的另一条α 2（I）带。当在脯氨酰和赖氨酰羟基化的抑制剂α α '-联吡啶存在下合成时，培养基和细胞层的前胶原和胶原含有正常和较慢的α 2（I），但仅含有正常的α 1（I）。两种形式的α 2（I）链的持续存在表明COL 1A 2基因发生了突变。胶原蛋白的CNBr裂解产生过度修饰的α 1（I）CB 3和CB 7肽，并延迟α 2（I）CB 3 -5肽的迁移。在α，α ′-联吡啶处理后也发现延迟的CB 3 -5。这些数据定位了α 2（I）（CB 3 -5）中aa 353和551之间的突变。对该区域的亚克隆等位基因进行测序，发现一个等位基因在nt 1671处发生G → A转换，将α 2（I）链中的Gly 421变为Asp。使用α 2（I）nt 1585处的常见G-->A多态性和罕见变体Arg 618-->Gln的存在，证明突变发生在父系来源的等位基因上（菲利普斯等人，1990），在父亲的基因组DNA和先证者的突变等位基因。前胶原处理正常。慢α 2（I）胶原蛋白的Tm比对照低2 ℃，表明三螺旋稳定性降低。突变型胶原被掺入到培养的成纤维细胞沉积的细胞外基质中，由于Arg->Gln变体仅引起轻微的电泳延迟，所以Gly->Asp取代必然引起α 2（I）电泳迁移率的显著延迟。即使在没有过度修饰的情况下，凝胶迁移率的显著延迟表明突变的α 2（I）链中存在扭结。分泌的成纤维细胞前胶原的旋转阴影电子显微镜证实了在含有甘氨酸取代的螺旋区域中存在扭结。胶原螺旋的扭结发生在没有二聚体形成的情况下。扭结可能干扰正常的螺旋折叠，以及胶原纤维与胶原和非胶原细胞外基质蛋白的相互作用。
Type I collagen synthesized by cultured skin fibroblasts was analyzed biochemically and molecularly to characterize the defect in a patient affected by lethal Osteogenesis Imperfecta. The SDS-Urea-PAGE of procollagen and collagen revealed a broad alpha 1(I) band, a normal alpha 2(I) and another alpha 2(I) band migrating equidistant between al and a2. When synthesized in the presence of alpha alpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylation, procollagen and collagen of media and cell layers contained both normal and slower alpha 2(I), but only normal alpha 1(I). The persistence of the two forms of alpha 2(I) chains suggested a mutation in a COL1A2 gene. CNBr cleavage of collagen yielded overmodified alpha 1(I) CB3 and CB7 peptides and delayed migration of the alpha 2(I) CB3-5 peptide. A delayed CB3-5 was also found after alpha,alpha'-dipyridyl treatment. These data localized the mutation between aa 353 and 551 in alpha 2(I) (CB3-5). Sequencing the subcloned alleles in this region revealed a G-->A transition at nt 1671 in one allele, changing Gly 421 to Asp in an alpha 2(I) chain. The mutation was demonstrated to occur on the paternally derived allele, using a common G-->A polymorphism at alpha 2(I) nt 1585 and by the presence of a rare variant, Arg618-->Gln (Phillips et al., 1990), in the paternal genomic DNA and the proband's mutant allele. Procollagen processing was normal. The T-m of the slow alpha 2(I) collagen was 2 degrees C lower than the control, indicating decreased triple helix stability. Mutant collagen was incorporated in the extracellular matrix deposited by cultured fibroblasts.The dramatic delay in alpha 2(I) electrophoretic mobility must be induced by the Gly-->Asp substitution, since the Arg-->Gln variant causes only mild electrophoretic delay. Substantial delay in gel mobility even in the absence of overmodification suggested the presence of a kink in the mutated alpha 2(I) chains. Rotary shadowing electron microscopy of secreted fibroblast procollagen confirmed the presence of a kink in the region of the helix containing the glycine substitution. The kinking of the collagen helix occurs in the absence of dimer formation. Kinking may interfere with normal helix folding, as well as with the interactions of collagen fibrils with the collagenous and non-collagenous extracellular matrix proteins.