Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth

Abstract

The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1–147), a central fragment (aa 148–204) denoted SKK-fragment and a C-terminal fragment (aa 205–262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.

The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling

“…Furthermore, a recent study by Boskey et al showed the C-and N-terminal fragments of OPN, in this study, derived from milk OPN to promote de novo HA formation. Conversely, a central fragment inhibited it as is similar to bone OPN (Boskey et al 2012). This highlights the importance of the post-translational fragmentation of OPN in determining its function.…”

“…This inhibitory role of OPN is confirmed further by analysis of the Opn knockout mouse that has increased mineral content and size, as shown by Fourier transform infrared spectroscopy analysis in two different lines of Opn K/K mice at two different ages (Boskey et al 2002). More specifically, it has recently been shown that the ASARM peptide of OPN inhibits ECM matrix mineralisation by binding to HA crystals (Addison et al 2010, Boskey et al 2012. Furthermore, a recent study by Boskey et al showed the C-and N-terminal fragments of OPN, in this study, derived from milk OPN to promote de novo HA formation.…”

Intrinsically disordered proteins and biomineralization

“…We know that if a protein can bind to the mineral acting as an epitaxial nucleator, it also could coat the mineral surface and regulate the size or shape of that mineral. This phenomenon has been seen recently in HA binding studies using peptides [16] and proteins [13]. It is also likely that the functions of proteins that control mineralization in multicellular organisms are redundant, both because the pattern of mineralization may differ in different tissues throughout the organism, and because mineralization is so important for life itself.…”

Role of Osteopontin in Bovines and their Association with Different Traits: A Review

“…OPN functionally similar OPN iso forms are cell and tissue-specific and OPN expressed by different cell-types or under different conditions have been shown to be functionally different [13,14]. OPN is highly phosphorylated in milk and contains 25 phosphates distributed over 36 potential sites in the human milk OPN [14,15] and 22 phosphates and 28 potential sites in bovine milk OPN [16]. Bovine milk OPN contains three O-glycosylated threonine residues close to the integrin binding motifs [5].…”