Lightfoot S J, Holmes D F, Brass A, Grant M E, Byers P H, Kadler K E
Department of Biochemistry and Molecular Biology, University of Manchester, United Kingdom.
J Biol Chem. 1992 Dec 15;267(35):25521-8.
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.
I型前胶原是从4名患有II型成骨不全症（OI）的个体的真皮成纤维细胞培养基中纯化得到的，这些个体的I型前胶原COL1A1基因发生了突变。这些前胶原是正常分子与分子的混合物，在分子的一条或两条α1（I）链中，存在甘氨酸97被天冬氨酸取代、甘氨酸550被精氨酸取代、甘氨酸718被半胱氨酸取代以及甘氨酸883被天冬氨酸取代的情况。与对照I型前胶原相比，这些前胶原被前胶原N蛋白酶切割得更慢。初始相对速度与初始底物浓度的双倒数图表明，由于Vmax降低而非Km增加，OI前胶原均被N蛋白酶缓慢切割。这表明N蛋白酶对OI前胶原的缓慢切割是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α1（I）883 OI前胶原的脊椎动物胶原酶A片段，与样品中的正常脊椎动物胶原酶A片段相比，被N蛋白酶切割得也更慢。这些数据首次表明，从一个大体积残基取代甘氨酸的位点到前胶原分子的氨基末端，改变的三螺旋结构得以传播，并破坏了N蛋白酶切割位点的构象。在甘氨酸α1（I）-718被半胱氨酸取代的制剂中，分子的旋转阴影电子显微镜观察显示，在大约5%的分子群体中存在一个扭结，其中60%的分子异常，20%的分子含有二硫键。相比之下，通过旋转阴影电子显微镜观察，含有甘氨酸被天冬氨酸和精氨酸取代的前胶原与对照样品中的前胶原没有区别。此处的结果证实了先前的推测，即I型胶原α1（I）链中甘氨酸被半胱氨酸取代可在取代位点附近引入一个扭结。然而，扭结的存在并非N蛋白酶对异常前胶原延迟切割的先决条件。
