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Results for “Matrix Gla Protein”

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At least 19 recordsLinked to original sources

PubMed1998

Molecular cloning of avian matrix Gla protein.

Matrix Gla protein plays an essential role in preventing the calcification of blood vessel walls, cartilage and other tissues. We report here the primary structure of chicken matrix Gla protein as deduced from the cDNA sequence. The avian protein exhibited the characteristic motifs previously identified in the mammalian proteins, but its amino acid sequence shared only 51-56% identity with the latter proteins. Moreover, a region proposed to function as binding site for gamma-carboxylase in the mammalian proteins was poorly conserved in the chicken protein. Our sequence data should be helpful in the design of mutational analyses which are intended to characterize functional interactions of matrix Gla proteins with other proteins.

Amino Acid Sequence↗

PubMed1991

Vitamin K-dependent carboxylase: utilization of decarboxylated bone Gla protein and matrix Gla protein as substrates.

The ability of des-gamma-carboxy bone Gla protein (dBGP) and des-gamma-carboxy matrix Gla protein (dMGP) to act as substrates for the rat liver vitamin K-dependent carboxylase has been investigated. An amino-terminal 'propeptide' is present on the intracellular form of BGP and is thought to interact with a recognition site on the enzyme. dBGP, lacking this extension, is a poor, high apparent Km, carboxylase substrate, but is a much better substrate when free propeptide is added. MGP lacks an amino-terminal propeptide, but contains a a homologous region in the mature protein. dMGP is an excellent substrate for the carboxylase with a low apparent Km and its carboxylation is inhibited by free propeptide.

Amino Acid Sequence↗

PubMed1983

Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone.

A new protein has been isolated from CaCl2/urea extracts of demineralized bovine bone matrix. This protein has five to six residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid (Gla), and we have accordingly designated it matrix Gla protein. Matrix Gla protein is a 15,000 dalton protein whose amino acid composition includes a single disulfide bond. The absence of 4-hydroxyproline in matrix Gla protein demonstrates that it is not a precursor to bone Gla protein, 5,800 dalton protein which has a residue of 4-hydroxyproline at position 9 in its sequence. Matrix Gla protein also does not cross-react with antibodies raised against bone Gla protein.

1-Carboxyglutamic Acid↗

PubMed1988

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase.

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.

Animals↗

PubMed2001

Total chemical synthesis of human matrix Gla protein.

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five gamma-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 microg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in alpha-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.

Blotting, Western↗

PubMed1990

Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene.

Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors.

Amino Acid Sequence↗

PubMed1991

Chromosomal assignment in mouse of matrix Gla protein and bone Gla protein genes.

Matrix Gla protein (MGLAP) and bone Gla protein (BGLAP) are calcium-binding, vitamin K-dependent proteins produced by cells of the osteoblastic lineage. Sequence homology suggests that the genes for these proteins evolved from a common ancestor. Somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map Mglap to mouse Chromosome 6 and Bglap to mouse Chromosome 3. Human MGLAP has previously been mapped to chromosome 12p, a region with homology to mouse Chromosome 6, and human BGLAP has been mapped to chromosome 1q, a region with homology to mouse Chromosome 3. It appears that BGLAP is the third calcium-binding protein that maps to human chromosome 1q and mouse Chromosome 3.

1-Carboxyglutamic Acid↗

PubMed1994

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix GLA protein in vitro.

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, gamma-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 micrograms/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes.

Animals↗

PubMed1998

Isolation and radiation hybrid mapping of dinucleotide repeat polymorphism at the human matrix Gla protein (MGP) locus.

Matrix Gla protein (MGP) is an 84-residue, vitamin K-dependent protein expressed by chondrocytes and vascular smooth muscle cells, and is a potent regulator of calcium deposition in cartilage and arterial wall. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human MGP gene. This polymorphism will be useful in genetic studies of arteriosclerosis and osteoporosis.

Alleles↗

PubMed1992

Expression of the matrix Gla protein in urogenital malignancies.

Matrix Gla protein (MGP), is a vitamin-K-dependent protein which is synthesized in a variety of tissues such as lung, heart, kidney, cartilage and bone. The function of MGP in these tissues is unclear. We have previously reported elevated MGP mRNA levels in a breast-cancer cell line, 600PEI, as compared to normal breast epithelium. Here we describe high MGP expression in primary renal-cell carcinomas, prostate carcinomas and testicular germ-cell tumors, as determined by Northern analysis. MGP was over-expressed in 21 out of 28 patients with renal-cell carcinoma, and in 16 out of 29 patients with testicular germ-cell tumors, as compared to matched normal tissues. For the renal-cell carcinomas, a statistically significant inverse correlation was observed between the level of MGP expression and tumor size, lymph-node metastasis and tumor grade. MGP was also highly expressed in 13 primary prostatic carcinomas as compared to prostate cell lines derived from metastatic tumors, and to lymph-node metastasis. Our findings indicate that the loss of MGP expression may be associated wih tumor progression and metastasis.

Carcinoma, Renal Cell↗

PubMed2000

Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2).

Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. As the first step to test this hypothesis, we demonstrate that MGP is a binding protein for 125I-BMP-2. Optimal binding is dependent on metals which suggests that the metal binding Gla region in MGP is involved. MGP is shown to undergo a Ca++ induced conformational change despite the presence of the gamma-carboxylase binding site being part of the mature protein sequence. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. Antibodies were used in an attempt to identify MGP in bovine serum. Conformational specific MGP antibodies were shown to also recognize the Gla region in prothrombin and factor X but did not identify MGP in serum. This finding is supported by electrophoresis data which demonstrate the absence of MGP among Ba-citrate absorbed vitamin K-dependent serum proteins. We conclude that MGP does not exist in normal bovine serum.

Animals↗

PubMed1998

Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression.

The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa's method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers alpha-SM actin, SM22alpha, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA. In contrast, OP mRNA was barely detectable in calcified human VSMCs and pericyte nodules, nor was OP protein detected, suggesting that OP was not necessary for calcification to occur. These studies reveal that human VSMCs are capable of inducing calcification and that MGP may have a role in human vascular calcification.

Adolescent↗

PubMed2001

Down-regulation of matrix Gla protein messenger RNA in human colorectal adenocarcinomas.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein commonly found in a variety of tissues. In this study, we describe the potential use of MGP gene expression as the tumor marker of colorectal cancer. A decrease in expression of the MGP gene was also discovered in colorectal cancer using differential screening of cDNA libraries. The MGP expression in 80 human colorectal adenocarcinomas was quantified by a Northern blot analysis to better define the expression pattern of MGP in colorectal cancer. The expression of MGP mRNA was reduced in 63 of 80 (79%) colorectal adenocarcinomas (P<0.001) as compared to the mRNA in adjacent normal tissue, implying that a decrease in MGP expression is associated with colorectal cancer development. The proportion of tumors with downregulated expression of MGP was lower in Duke's A/B than Duke's C/D (34 of 47 versus 26 of 33, respectively) tumors and was lower in moderate differentiation than poor differentiation (44 of 64 versus 16 of 16, respectively). However, chi(2) analysis does not reveal any correlation between a loss of MGP expression and tumor progression or differentiation state. In conclusion, the downregulation of MGP mRNA generally occurs in colorectal adenocarcinomas. Although the role of MGP in cancer development is unknown, the reduced expression of MGP may be used to distinguish the normal colorectal cells from malignant cells.

Adenocarcinoma↗

PubMed1991

Developmental expression and hormonal regulation of the rat matrix Gla protein (MGP) gene in chondrogenesis and osteogenesis.

Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintenance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed: an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25-(OH)2D3 (10(-8) M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post-transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintenance of a collagenous matrix as reflected by increases in MGP mRNA during these periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

Identification of a novel negative retinoic acid responsive element in the promoter of the human matrix Gla protein gene.

The vitamin K-dependent matrix Gla protein (MGP) is synthesized in a wide variety of tissues such as lung, heart, kidney, cartilage, and bone. Expression of the MGP gene is regulated by various growth factors, steroid hormones, and the vitamin A metabolite retinoic acid (RA). In this report, we present evidence that RA down-regulates MGP gene expression in different rat and human cell lines via endogenous retinoid receptors [RA receptor (RAR) and retinoid X receptor (RXR)]. Repression of the human MGP (hMGP) gene is specifically mediated by ligand-activated RAR and RXR. Deletion analysis led to the identification of a novel negative response element (NRE) within the hMGP promoter. DNA binding studies performed with bacterially expressed RAR/RXR reveal the formation of a specific heterodimer/NRE complex. Furthermore, electrophoretic mobility-shift assays performed with proteins from RA-treated cells show that endogenous RAR/RXR binds to the NRE. We demonstrate that the NRE contains a CCAAT box and that both RAR/RXR and CCAAT-binding proteins such as c/EBP beta recognize this common regulatory sequence in the hMGP promoter. Our results indicate that RA-mediated repression of the hMGP gene is due to binding of liganded RAR/RXR to a novel negative RA response element.

Animals↗

PubMed1997

Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.

Calcification of the extracellular matrix (ECM) can be physiological or pathological. Physiological calcification occurs in bone when the soft ECM is converted into a rigid material capable of sustaining mechanical force; pathological calcification can occur in arteries and cartilage and other soft tissues. No molecular determinant regulating ECM calcification has yet been identified. A candidate molecule is matrix GLA protein (Mgp), a mineral-binding ECM protein synthesized by vascular smooth-muscle cells and chondrocytes, two cell types that produce an uncalcified ECM. Mice that lack Mgp develop to term but die within two months as a result of arterial calcification which leads to blood-vessel rupture. Chondrocytes that elaborate a typical cartilage matrix can be seen in the affected arteries. Mgp-deficient mice additionally exhibit inappropriate calcification of various cartilages, including the growth plate, which eventually leads to short stature, osteopenia and fractures. These results indicate that ECM calcification must be actively inhibited in soft tissues. To our knowledge, Mgp is the first inhibitor of calcification of arteries and cartilage to be characterized in vivo.

Animals↗

PubMed1996

Matrix Gla protein gene expression is elevated during postnatal development.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein with a wide tissue distribution. Developmental expression of the MGP gene is characterized by competitive RT-PCR in kidney and calvaria. High levels of MGP mRNA were observed in kidneys and calvaria from 19-day-old embryos to 1-month-old rats. There was a peak in MGP mRNA at 7 days in both tissues. MGP mRNA expression was very low or undetectable in 3-, 5- and 7- month-old kidneys. Similar observations were seen in lung, heart and spleen. However, in connective tissues like calvaria, tibia and trachea, low levels of MGP mRNA are maintained throughout life. Kidney MGP protein was present from birth to 15 days, with the highest MGP protein level at 7 days. Calvarial MGP protein was present throughout development and maturation but peaked at 7 days. The highest MGP protein levels were coincident with peak levels of MGP mRNA. Thus, MGP protein level correlated with mRNA level during rat development. In situ hybridization revealed that MGP staining was most intense in the straight tubules of the developing kidney medulla at 7 days. Staining was absent in stromal cells and in mature nephrons. Taken together, our finding of high MGP mRNA and its intense in situ staining during the postnatal growth phase prove that increased MGP synthesis occurs at a specific time and place during development and maturation.

Animals↗

PubMed1993

Matrix Gla protein mRNA expression in cultured type II pneumocytes.

Matrix Gla protein (MGP) was first isolated from the matrix fraction of bone. This highly conserved vitamin K-dependent protein of 14 kDa has been identified in numerous tissues and cells, and its mRNA was recently found to be abundant in rat lung. Relatively low MGP protein levels in many soft tissues where its mRNA is high suggests an important secretory function for this protein. We have found a high specific activity of vitamin K-dependent carboxylase in microsomes of rat pulmonary type II cells and the presence of numerous endogenous substrates, including one of 13-15 kDa. To investigate the possibility that MGP and its mRNA could be localized in type II cells, rat MGP and actin cDNA probes were hybridized to total RNA obtained from freshly isolated type II cells and from cells cultured for up to 6 days. MGP mRNA increased 5- to 6-fold relative to beta-actin mRNA from days 3 to 6 in primary culture and MGP secretion increased nearly 60-fold during that interval. MGP mRNA and MGP secretion decreased 25-75% if cultures were supplemented with vitamin K quinone. Vitamin K deficiency, caused by carbon stripping the serum or treatment of cell cultures with warfarin, resulted in an induction of carboxylase activity and elevated MGP mRNA. In parallel experiments, carboxylase specific activity also increased during culture in the presence or absence of vitamin K. Retinoic acid further increased steady-state mRNA levels and MGP secretion at later culture intervals, an effect which was serum dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗