Price P A, Fraser J D, Metz-Virca G
Department of Biology, University of California, San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1987 Dec;84(23):8335-9. doi: 10.1073/pnas.84.23.8335.
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.
基质γ-羧基谷氨酸蛋白（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依赖型氨基酸γ-羧基谷氨酸（Gla）残基。我们利用针对MGP产生的抗体和寡核苷酸探针，筛选了一个由经1α,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λgt11 cDNA文库。通过对几个克隆的cDNA进行测序，我们确定了一个523个碱基对的序列，该序列预测了一个84个残基的成熟MGP和一个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末端还有5个额外的残基（-Arg-Arg-Gly-Ala-Lys），这在从牛骨中分离的MGP序列中未见。大鼠MGP的结构为深入了解维生素K依赖型γ-羧化酶识别底物的机制提供了线索。目前的研究表明，与其他维生素K依赖型蛋白质不同，MGP缺乏前肽。MGP前肽的缺失表明，维生素K依赖型蛋白质的γ-羧化和分泌不一定与前肽的存在或其蛋白水解去除有关。其他维生素K依赖型蛋白质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γ-羧化酶识别底物很重要。成熟的MGP有一个与其他维生素K依赖型蛋白质的前肽同源的序列片段（第15 - 30位残基），可能在γ-羧化酶识别中起相同作用。大鼠MGP还有第二个序列，最近在所有已知的维生素K依赖型脊椎动物蛋白质中都已鉴定出，即不变单元Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys（EXXXEXC）。由于该单元中的谷氨酸残基是γ-羧化的位点，有人提出EXXXEXC单元可能使γ-羧化酶能够区分底物和产物。维生素K依赖型蛋白质共有的两种结构，即同源前肽结构域和不变的EXXXEXC单元存在于成熟MGP中，这表明去γ-羧基-MGP应该是一种用于分析底物识别和产物解离机制的优秀体外γ-羧化酶底物。
