Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification

J Thromb Haemost. 2007 Dec;5(12):2503-11. doi: 10.1111/j.1538-7836.2007.02758.x. Epub 2007 Sep 10.

Abstract

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.

Methods and 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Carboxyglutamic Acid / metabolism
  • Adolescent
  • Adult
  • Amino Acid Sequence
  • Calcinosis / metabolism
  • Calcinosis / prevention & control*
  • Calcium / metabolism*
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Extracellular Matrix Proteins / chemistry
  • Extracellular Matrix Proteins / metabolism*
  • Female
  • Humans
  • Male
  • Middle Aged
  • Molecular Sequence Data
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / metabolism*
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism*
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Protein Structure, Tertiary
  • Serine / metabolism
  • Transport Vesicles / metabolism
  • Vitamin K / antagonists & inhibitors
  • Vitamin K / metabolism
  • Vitamin K 1 / pharmacology
  • Warfarin / pharmacology

Substances

  • Calcium-Binding Proteins
  • Extracellular Matrix Proteins
  • Peptide Fragments
  • matrix Gla protein
  • Vitamin K
  • Serine
  • 1-Carboxyglutamic Acid
  • Warfarin
  • Vitamin K 1
  • Calcium