Periostin, a Member of a Novel Family of Vitamin K-dependent Proteins, Is Expressed by Mesenchymal Stromal Cells

Abstract

The modification of glutamic acid residues to γ-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme γ-glutamylcarboxylase. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, βig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent γ-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro. The modification of glutamic acid residues to γ-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme γ-glutamylcarboxylase. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, βig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent γ-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro. Bone marrow stroma contains a population of adherent cells referred to as mesenchymal stromal cells (MSCs), 4The abbreviations used are: MSC, marrow-derived mesenchymal stromal cells; Gla, γ-carboxyglutamic acid; CM, conditioned medium; ECM, extracellular matrix; PN, periostin; OC, osteocalcin; MGP, matrix Gla protein; MS, mass spectrometry; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, h