Weis M A, Wilkin D J, Kim H J, Wilcox W R, Lachman R S, Rimoin D L, Cohn D H, Eyre D R
Orthopaedic Research Laboratories, University of Washington, Seattle, Washington 98195-6500, USA.
J Biol Chem. 1998 Feb 20;273(8):4761-8. doi: 10.1074/jbc.273.8.4761.
Type II collagen mutations have been identified in a phenotypic continuum of chondrodysplasias that range widely in clinical severity. They include achondrogenesis type II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal dysplasia, Kniest dysplasia, and Stickler syndrome. We report here results that define the underlying genetic defect and consequent altered structure of assembled type II collagen in a neonatal lethal form of Kniest dysplasia. Electrophoresis of a cyanogen bromide (CNBr) (CB) digest of sternal cartilage revealed an alpha1(II)CB11 peptide doublet and a slightly retarded mobility for all major CB peptides, which implied post-translational overmodification. Further peptide mapping and sequence analysis of CB11 revealed equal amounts of a normal alpha1(II) sequence and a chain lacking the 18 residues (361-378 of the triple helical domain) corresponding to exon 24. Sequence analysis of an amplified genomic DNA fragment identified a G to A transition in the +5 position of the splice donor consensus sequence of intron 24 in one allele. Cartilage matrix analysis showed that the short alpha1(II) chain was present in collagen molecules that had become cross-linked into fibrils. Trypsin digestion of the pepsin-extracted native type II collagen selectively cleaved the normal length alpha1(II) chains within the exon 24 domain. These findings support a hypothesis that normal and short alpha-chains had combined to form heterotrimeric molecules in which the chains were in register in both directions from the deletion site, accommodated effectively by a loop out of the normal chain exon 24 domain. Such an accommodation, with potential overall shortening of the helical domain and hence misalignment of intermolecular relationships within fibrils, offers a common molecular mechanism by which a group of different mutations might act to produce the Kniest phenotype.
在临床严重程度差异很大的一系列软骨发育不良中已鉴定出II型胶原突变。它们包括II型软骨发育不全、低软骨发育不全、先天性脊柱骨骺发育不良、脊柱干骺端发育不良、克尼斯特发育不良和斯蒂克勒综合征。我们在此报告了一些结果，这些结果确定了新生儿致死型克尼斯特发育不良中潜在的遗传缺陷以及组装后的II型胶原结构的改变。胸骨软骨的溴化氰（CNBr）消化产物的电泳显示出α1（II）CB11肽双峰，并且所有主要的CB肽迁移率略有延迟，这意味着存在翻译后过度修饰。对CB11进行进一步的肽图谱分析和序列分析发现，正常的α1（II）序列与缺少对应于外显子24的18个残基（三螺旋结构域的361 - 378位）的链的量相等。对扩增的基因组DNA片段进行序列分析，在一个等位基因中发现内含子24的剪接供体共有序列的+5位置发生了G到A的转变。软骨基质分析表明，短的α1（II）链存在于已交联成纤维的胶原分子中。用胃蛋白酶提取的天然II型胶原经胰蛋白酶消化后，可选择性地切割外显子24结构域内正常长度的α1（II）链。这些发现支持了这样一种假说，即正常和短的α链结合形成了异源三聚体分子，其中这些链在缺失位点的两个方向上都是对齐的，由正常链外显子24结构域的一个环有效容纳。这种容纳方式可能导致螺旋结构域整体缩短，从而使纤维内分子间关系错位，为一组不同的突变产生克尼斯特表型提供了一种共同的分子机制。
