Phosphorylation-dependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage

Abstract

The SIBLING family (small integrin-binding ligand N-linked glycoproteins) of mineral-regulating proteins, which includes matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), contains an acidic serine-and aspartate-rich motif (ASARM). Xlinked hypophosphatemia caused by inactivating mutations of the PHEX gene results in elevated mineralization-inhibiting MEPE-derived ASARM peptides. Although the OPN ASARM motif shares 60% homology with MEPE ASARM, it is still unknown whether OPN ASARM similarly inhibits mineralization. In this study we have examined the role of OPN ASARM and its interaction with PHEX enzyme using an osteoblast cell culture model, mass spectrometry, mineral-binding assays, and computational modeling. MC3T3-E1 osteoblast cultures were treated with differently phosphorylated OPN ASARM peptides [with 5 phosphoserines (OpnAs5) or 3 phosphoserines (OpnAs3)] or with control nonphosphorylated peptide (OpnAs0). Phosphorylated peptides dose-dependently inhibited mineralization, and binding of phosphorylated peptides to mineral was confirmed by a hydroxyapatite-binding assay. OpnAs0 showed no binding to hydroxyapatite and did not inhibit culture mineralization. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy with increasing phosphorylation consistent with hydroxyapatite-binding experiments and inhibition of culture mineralization. Addition of PHEX rescued inhibition of mineralization by OpnAs3. Mass spectrometry of cleaved peptides after ASARM-PHEX incubations identified OpnAs3 as a PHEX substrate. We conclude that OPN ASARM inhibits mineralization by binding to hydroxyapatite in a phosphorylation-dependent manner and that this inhibitor can be cleaved by PHEX, thus providing a mechanistic explanation for how loss of PHEX activity in X-linked hyposphosphatemia can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia. ß

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

“…(13) These findings align with the concept that proteolytic processing of OPN and its peptides by PHEX contribute to the local control of crystal growth in bone mineralization. The selectivity of PHEX hydrolysis for OPN degradation relative to other acidic SIBLING proteins was assessed using another phosphorylated protein-BSP-also thought to play a role in bone and tooth mineralization and which in vitro acts as a potent nucleator of mineralization.…”

“…Phosphorylated full-length OPN is a potent mineralization inhibitor of osteoblast cell cultures, (19) as are its phosphorylated peptides, (13,15) with some evidence for an inhibitory function in bone in vivo. (34) Thus, both the full-length protein and its peptides appear to be important in the inhibition of mineralization.…”

“…As part of this process, enzymatic activity may be further controlled by enzymeinhibiting interactions, such as that observed between PHEX and glycosaminoglycans, (36) or by temporal expression of PHEX. (53,62) Given the potent mineralization-inhibiting activity of OPN and its phosphorylated peptide motifs such as the ASARM region, (13,14) it is reasonable to consider the notion that the required, extensive/complete degradation of OPN by PHEX does not occur in XLH, thus contributing to the osteomalacic phenotype.…”

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

“…The different aspects of collagen phosphorylation on processing, fibril formation or protein interactions have recently been reviewed (46). Other noncollagenous extracellular matrix proteins are also crucial in bone and tooth formation via the control of hydroxyapatite crystallization (47,48). These secretory calcium-binding phosphoproteins (osteopontin, OPN; dentin matrix protein-1, DMP1; bone sialoprotein, BSP; matrix extracellular phosphoglycoprotein, MEPE and dentin sialophosphoprotein, DSPP) belong to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and are heavily phosphorylated (49).…”

Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

“…It was recently demonstrated that osteopontin (OPN), a member of the small integrin-binding ligand N-linked glycoproteins family, is also a source of ASARM peptide. Phosphorylated ASARM peptide derived from OPN was able to inhibit mineralization in osteoblast cultures and the addition of PHEX rescues this effect (Addison et al 2010). Consequently, a possible dual mechanism for PHEX has been suggested: to prevent the release of ASARM peptides from proteins like MEPE and OPN and to promote ASARM's degradation.…”