Proline and γ-Carboxylated Glutamate Residues in Matrix Gla Protein Are Critical for Binding of Bone Morphogenetic Protein-4

Abstract

Abstract-Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP).MGP binds calcium ions through ␥-carboxylated glutamates (Gla residues) and inhibits bone morphogenetic protein (BMP)-2/-4. We hypothesized that a conserved proline (Pro)64 is essential for BMP inhibition. We further hypothesized that calcium binding by the Gla residues is a prerequisite for BMP inhibition. Site-directed mutagenesis was used to modify Pro64 and the Gla residues, and the effect on BMP-4 activity, and binding of BMP-4 and calcium was tested using luciferase reporter gene assays, coimmunoprecipitation, crosslinking, and calcium quantification. The results showed that Pro64 was critical for binding and inhibition of BMP-4 but not for calcium binding. The Gla residues were also required for BMP-4 binding but flexibility existed. As long as 1 Gla residue remained on each side of Pro64, the ability to bind and inhibit BMP-4 was preserved. Chelation of calcium ions by EDTA or warfarin treatment of cells led to loss of ability of MGP to bind BMP-4. Our results also showed that phenylalanine could replace Pro64 without loss of function and that zebrafish MGP, which lacks upstream Gla residues, did not function as a BMP inhibitor. 1 Patients with defined mutations in the MGP gene (Keutel syndrome) have widespread calcification in the tracheobronchial tree, and the quality and duration of life appears to be determined by the extent of pulmonary disease. 2,3 We and others have identified MGP as an inhibitor of bone morphogenetic protein (BMP). 4 -7 Specifically, we have shown that MGP binds and inhibits BMP-2 and -4, which are both detected in calcified atherosclerotic lesions. 8 -11 We have also shown that MGP-deficient cells have increased susceptibility to BMP-2-induced cell differentiation 12 and that MGP interferes with BMP-4 activity in the developing lungs of MGP transgenic mice. 13 MGP is known to be a calcium-binding protein. Calcium is bound through ␥-carboxylated glutamates, so-called "Gla residues," and induces conformational changes in the MGP protein. 4,14 Warfarin interferes with the vitamin K-dependent ␥-carboxylation and renders MGP nonfunctional, 15 presumably through loss of calcium binding. The majority of the Gla residues are located in the center of the MGP protein (amino acid residue 56 to 71 in human MGP), a region that has been implicated in the binding of BMP. 4 However, the precise amino acid residues involved in BMP binding have not been identified.In the BMP type I receptor, a phenylalanine residue (Phe85) plays a key role in binding BMP-2. 16 The aromatic side chain of Phe85, with "knob-into-hole" packing, points into a hydrophobic pocket formed at the interfaces of 2 BMP-2 monomers. Similarly, a critical proline residue (Pro35) in the BMP inhibitor Noggin occupies the same pocket, 17,18 thereby preventing ligand-receptor binding. When examining the protein sequence of human MGP, we found a conserved proline residue (Pro64) centrally located in the BMP-bin...

Reducing Jagged 1 and 2 levels prevents cerebral arteriovenous malformations in matrix Gla protein deficiency

“…The functionality of MGP depends on correct gammacarboxylation of glutamate residues in the MGP protein (14), and non-gamma-carboxylated MGP has been observed in the vasculature after warfarin treatment (31). If there is sufficient inhibition of the gamma-carboxylation, it could increase the risk of AVM formation, although this has not been investigated.…”

“…In turn, activation of ALK1 by its ligand BMP-9 induces expression of matrix Gla protein (MGP), an inhibitor of BMP-2, -4 and -7 (12)(13)(14)(15), which is highly expressed in brain, lungs, and kidneys (13,16). MGP provides negative feedback inhibition for BMP-4 and -7 (13,17), which limits the ALK1 induction.…”

Vascular Calcification

“…100 -102 MGP binds BMP-2 to mediate the osteoblastic differentiation of VSMCs and inhibits the activity of BMP-2 in the differentiation of mesenchymal cells. 103 Furthermore, MGP expression is downregulated and osteoblastic markers such as collagen type II and OC are upregulated in calcified vessels. MGP also binds Ca 2ϩ crystals and inhibits crystal growth.…”

Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium

“…21,22 CV2 (100 ng) was mixed with BMP-9 (50 ng) or BMP-4 (50 ng), chemically cross-linked with disuccinimidyl suberate (DSS) and the cross-linked products were analyzed by immunoblotting as described in the next section. In some experiments, up to 20-fold excess of BMP-4 was used, which corresponded to 1000 ng of BMP-4.…”