Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.
Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.
Wajih
Nadeem
N
Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27517, USA.
Borras
Terete
T
Xue
Wei
W
Hutson
Susan M
SM
Wallin
Reidar
R
D000256:Adenoviridae / Q000235:genetics; D001158:Arteries / Q000378:metabolism; D001692:Biological Transport; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D047388:Casein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008861:Microsomes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D010766:Phosphorylation; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000737:chemistry; D000097568:Matrix Gla Protein
D001710:Biotin; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D002135:Calcium-Binding Proteins; D047388:Casein Kinases; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D016212:Transforming Growth Factor beta; D000509:alpha-Fetoproteins; D000097568:Matrix Gla Protein; C525718:BMP2 protein, human