PB1252 - Role of matrix Gla protein in vascular calcification: hard chemistry for soft vessels

Background: Vascular calcification is implicated in pathologies such as atherosclerosis and chronic kidney disease. However, much is unclear regarding initiation and inhibition of calcification while detection can only be achieved at a late stage, hampering intervention. A key player in this process is matrix Gla protein (MGP), a heavily post-translationally modified inhibitor of calcification. While MGP is recognized as one of the strongest inhibitors of ectopic calcification, its mechanism of action is unknown. Further research into MGP’s function is hampered by the inability in obtaining homogenously modified variants of MGP.

Aims: To study intra- and extracellular structure-function relationships of different variants of MGP containing combinations of serine-phosphorylation and glutamate-γ-carboxylation.

Methods: We have employed a 3-fragment, one-pot, chemical protein synthesis approach to synthesize all possible combinations of post-translationally modified variants of MGP (unmodified, phosphorylated, carboxylated, phosphorylated-carboxylated). These variants were tested for their ability to inhibit in vitro formation of calcium phosphate deposits. Cellular effects involved with calcification were evaluated in a vascular calcification assay where MGP variants were added to human vascular smooth muscle cells (hVSMC) cultured under calcifying conditions.

Results: Our chemical protein synthesis approach has provided access to diverse homogenous post-translationally modified variants of MGP offering unique tools for research of MGP’s structure-function relationships. First results of biological evaluation showed that unmodified MGP is unable to interfere with calcium phosphate precipitation whereas all post-translationally modified variants showed a dose-dependent inhibitory effect. Moreover, we could show a dose-dependent inhibitory effect of post-translationally modified MGP on calcifying hVSMCs.

Conclusion(s): Results of both the non-cellular and cellular vascular calcification model assays confirm importance of glutamate-γ-carboxylation but also show a clear effect of serine-phosphorylation on inhibition of calcium crystal formation. Currently, we investigate effects of addition of different MGP variants on hVSMC differentiation and phenotype switching.

Project is funded by the Dutch Heart Foundation (2019T013) to SMA