Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase.
Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase.
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DOI：
10.1073/pnas.84.23.8335
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发表时间：
1987-12
影响因子：
11.1
通讯作者：
Paul A. Price;James D. Fraser;G. Metz-Virca
中科院分区：
文献类型：
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作者：
Paul A. Price;James D. Fraser;G. Metz-Virca
基质 Gla 蛋白 (MGP) 是一种存在于骨骼、牙本质和软骨中的低分子量蛋白质，含有 5 个维生素 K 依赖性氨基酸 γ-羧基谷氨酸 (Gla) 残基。我们使用针对 MGP 的抗体和寡核苷酸探针来筛选由已用 1 α,25-二羟基维生素 D3 预处理的大鼠骨肉瘤细胞（ROS 17/2 系）构建的 lambda gt11 cDNA 文库。通过对几个克隆的 cDNA 进行测序，我们建立了一个 523 个碱基对的序列，可预测 84 个残基的成熟 MGP 和 19 个残基的疏水信号肽。从cDNA序列预测的84个残基的成熟大鼠MGP在其C末端具有额外的5个残基(-Arg-Arg-Gly-Ala-Lys)，这在从牛骨分离的MGP的序列中未见。大鼠 MGP 的结构提供了对维生素 K 依赖性 γ-羧化酶识别底物的机制的深入了解。目前的研究表明，与其他维生素 K 依赖性蛋白质不同，MGP 缺乏前肽。 MGP 前肽的缺失表明，维生素 K 依赖性蛋白的 γ-羧化和分泌不需要与前肽的存在或其蛋白水解去除相关。其他维生素 K 依赖性蛋白的前肽在结构上是同源的，并且有证据表明这种同源前肽结构域对于 γ-羧化酶的底物识别很重要。成熟的 MGP 具有与其他维生素 K 依赖性蛋白的前肽同源的序列片段（残基 15-30），并且可能在 γ-羧化酶识别中发挥相同的作用。大鼠 MGP 还具有最近在所有已知的维生素 K 依赖性脊椎动物蛋白中发现的第二个序列，即不变单位 Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys (EXXXEXC)。由于该单元中的谷氨酸残基是 γ-羧化位点，因此有人建议 EXXXEXC 单元可以允许 γ-羧化酶区分底物和产物。成熟 MGP 中存在维生素 K 依赖性蛋白共有的两种结构，即同源前肽结构域和不变的 EXXXEXC 单元，这表明 des-gamma-羧基-MGP 应该是一种出色的体外 gamma-羧化酶底物，用于分析底物识别和产物解离所涉及的机制。
Matrix Gla protein (MGP), a low molecular weight protein found in bone, dentin, and cartilage, contains 5 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid (Gla). We have used antibodies raised against MGP and oligonucleotide probes to screen a lambda gt11 cDNA library constructed from the rat osteosarcoma cells (line ROS 17/2) that had been pretreated with 1 alpha,25-dihydroxyvitamin D3. By sequencing several cloned cDNAs, we established a 523-base-pair sequence that predicts an 84-residue mature MGP and a 19-residue hydrophobic signal peptide. The 84-residue mature rat MGP predicted from the cDNA sequence has an additional 5 residues at its C terminus (-Arg-Arg-Gly-Ala-Lys) not seen in the sequence of MGP isolated from bovine bone. The structure of rat MGP provides insight into the mechanisms by which the vitamin K-dependent gamma-carboxylase recognizes substrate. The present studies show that MGP, unlike other vitamin K-dependent proteins, lacks a propeptide. The absence of an MGP propeptide demonstrates that gamma-carboxylation and secretion of vitamin K-dependent proteins need not be linked to the presence of a propeptide or to its proteolytic removal. The propeptides of other vitamin K-dependent proteins are structurally homologous, and there is evidence that this homologous propeptide domain is important to substrate recognition by the gamma-carboxylase. Mature MGP has a sequence segment (residues 15-30) that is homologous to the propeptide of other vitamin K-dependent proteins and probably serves the same role in gamma-carboxylase recognition. Rat MGP also has a second sequence that has recently been identified in all known vitamin K-dependent vertebrate proteins, the invariant unit Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys (EXXXEXC). Since the glutamic residues in this unit are sites of gamma-carboxylation, it has been suggested that the EXXXEXC unit could allow the gamma-carboxylase to discriminate between substrate and product. The demonstration that two structures common to vitamin K-dependent proteins, the homologous propeptides domain and the invariant EXXXEXC unit, are in mature MGP indicates that des-gamma-carboxy-MGP should be an excellent in vitro gamma-carboxylase substrate for analysis of mechanisms involved in substrate recognition and product dissociation.