Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.
Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.
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DOI：
10.1016/s0021-9258(19)74071-x
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发表时间：
1992-12
期刊：
影响因子：
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通讯作者：
S. Lightfoot;D. Holmes;A. Brass;M. E. Grant;P. Byers;K. Kadler
中科院分区：
文献类型：
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作者：
S. Lightfoot;D. Holmes;A. Brass;M. E. Grant;P. Byers;K. Kadler
I 型原胶原从四位 II 型成骨不全 (OI) 患者的真皮成纤维细胞培养基中纯化，这些患者的 I 型原胶原 COL1A1 基因发生突变。前胶原是正常分子和在分子的一条或两条 α1 (I) 链中含有天冬氨酸取代甘氨酸、精氨酸取代甘氨酸 550、半胱氨酸取代甘氨酸 718 和天冬氨酸取代甘氨酸 883 的分子的混合物。前胶原 N-蛋白酶对前胶原的切割速度比对照 I 型前胶原慢。初始相对速度和初始底物浓度的双倒数图表明，由于 Vmax 降低而不是 Km 增加，OI 前胶原都被 N-蛋白酶缓慢裂解。这表明 N-蛋白酶对 OI 前胶原的缓慢裂解是 N-蛋白酶-前胶原复合物缓慢转化的结果。进一步的实验表明，含有 N-蛋白酶切割位点但不包含取代位点的甘氨酸 α 1(I) 883 OI 前胶原天冬氨酸的脊椎动物胶原酶 A 片段被 N-蛋白酶切割的速度也比样品中正常脊椎动物胶原酶 A 片段慢。这些数据首次表明，改变的三螺旋结构从甘氨酸的大残基取代位点传播到原胶原分子的氨基末端，并破坏了 N-蛋白酶切割位点的构象。在制备甘氨酸 α 1(I)-718 的半胱氨酸时，分子的旋转阴影电子显微镜显示，大约 5% 的分子群中存在扭结，其中 60% 是异常的，20% 含有二硫键。相比之下，通过旋转阴影电子显微镜无法将含有天冬氨酸和精氨酸甘氨酸的前胶原与对照样品中的前胶原区分开。这里的结果证实了之前的建议，即在 I 型胶原蛋白的 α1(I) 链中用半胱氨酸取代甘氨酸会在取代位点附近引入扭结。然而，扭结的存在并不是 N-蛋白酶延迟裂解异常前胶原的先决条件。
Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.