Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.
BACKGROUND
Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood.


RESULTS
We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures.


CONCLUSIONS
These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.
Schurgers
L J
LJ
Cardiovascular Research Institute Maastricht and VitaK, University of Maastricht, Universiteitssingel 50, Maastricht, The Netherlands. l.schurgers@bioch.unimaas.nl
Spronk
H M H
HM
Skepper
J N
JN
Hackeng
T M
TM
Shanahan
C M
CM
Vermeer
C
C
Weissberg
P L
PL
Proudfoot
D
D
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D002114:Calcinosis / Q000378:metabolism / Q000517:prevention & control*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D014812:Vitamin K / Q000037:antagonists & inhibitors / Q000378:metabolism; D010837:Vitamin K 1 / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology; D000097568:Matrix Gla Protein
D015055:1-Carboxyglutamic Acid; D002118:Calcium; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D010446:Peptide Fragments; D012694:Serine; D014812:Vitamin K; D010837:Vitamin K 1; D014859:Warfarin; D000097568:Matrix Gla Protein