Active sites of human MEPE-ASARM regulating bone matrix mineralization
Introduction
Bone extracellular matrix contains several members of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family including dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), integrin binding sialoprotein (IBSP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (secreted phosphoprotein 1, SPP1). All the genes encoding these proteins exhibit similar exon structure and are located on chromosome 4q21 in humans and chromosome 5q in mice, supporting the view that the SIBLINGs arose from the secretory calcium-binding phosphoprotein family by gene duplication (Bellahcene et al., 2008; Kawasaki and Weiss, 2006; Rowe, 2012). While SIBLINGs share relatively little sequence identity overall, several motifs are highly conserved across the members and species: e.g., the RGD (Arg-Gly-Asp) cell attachment sequence and post-translational modification motifs such as phosphorylation, N-glycosylation, and proteolysis sites (Rowe et al., 2000; Argiro et al., 2001). The biological functions of SIBLINGs are diverse, but reflecting their evolutionary history and motif structure, one important function is in biomineralization (Minamizaki and Yoshiko, 2015).
Amongst the SIBLINGs, the roles of MEPE−originally cloned from the tumor of a patient suffering from tumor-induced osteomalacia (TIO) (Rowe et al., 2000)−in bone formation and phosphate metabolism are complex. Mepe knockout mice exhibited significantly increased bone mass as a result of increased osteoblast number and activity (Gowen et al., 2003). Transgenic mice overexpressing MEPE under the control of a 2.3-kb fragment of the Col1a1 promoter support the view that MEPE is antiosteogenic (David et al., 2009). TIO shares biochemical and clinical features with inherited hypophosphatemic rickets/osteomalacia (e.g., X-linked hypophosphatemia, XLH) (Rowe et al., 2000; Argiro et al., 2001; Rowe, 1998), suggesting a common etiology of TIO and XLH. Recombinant human MEPE (rhMEPE) decreases serum phosphate levels in mice (Rowe et al., 2004), while there is no significant difference in serum phosphate levels in Mepe knockout mice versus wild-type (WT) mice (Gowen et al., 2003). Backcrossing Mepe-deficient mice to Hyp mice, a mouse XLH model that highly expresses MEPE (Argiro et al., 2001; Guo et al., 2002), failed to rescue the hypophosphatemia and skeletal defects (Liu et al., 2005).
Further insight came from studies on ASARM (acidic serine- and aspartate-rich motif), a MEPE cleavage product found in bones and the systemic circulation (Bresler et al., 2004). The anti-mineralization activity of synthetic nonphosphorylated ASARM (MEPE-nASARM) peptide (hereinafter, “peptide” was abbreviated) was initially uncovered in mouse 2T3 osteoblastic cell cultures (Rowe et al., 2004). Thereafter, three phosphorylated Ser (pSer) residues in ASARM (MEPE-pASARM) were shown to be necessary for its anti-mineralization effect (e.g., increased osteoid in mouse bones) (Rowe et al., 2005), mineralization defects in mouse bone marrow cell (Liu et al., 2007), MC3T3-E1 cell (Addison et al., 2008), and mouse chondrocyte/cartilage (Staines et al., 2012) cultures, and inhibition of hydroxyapatite crystal formation (Boskey et al., 2010). Similar results were obtained in the synthetic pASARM of SPP1 (SPP1-pASARM) in vitro (Addison et al., 2010). The accumulation of MEPE-ASARM with or without pSer in the osteoid is seen in Hyp mice, and not only MEPE-pASARM (Addison et al., 2008) but also SPP1-pASARM (Addison et al., 2010) is cleaved and/or inactivated by PHEX, a membrane endopeptidase whose mutations causes XLH.
There are currently few drugs available for treatment of mineralization-associated disorders of skeletal or extraskeletal tissues (e.g., vascular calcification or ossification of ligaments, skeletal muscles, etc). To extend previous work and determine whether MEPE-ASARM may function as an anti-mineralization factor, we sought to clarify the anti-mineralization active site(s) of ASARM by examining the effects of synthetic ASARM and relevant peptides with or without pSer residues in well-established osteoblast culture models.
Section snippets
Peptide fragment synthesis
The designed peptide fragments with or without pSer residues were synthesized based on human MEPE-derived ASARM and relevant peptide fragments (ID1-11; Toray Research Center, Kanagawa, Japan) (Table 1). The peptide fragment used as an antigen for generation of polyclonal antibodies (see below) contained an additional Cys in the N-terminus of ID1 (ID1+c) to allow crosslinking to BSA by using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), according to the manufacturer's instructions. To
Results
To determine which site(s) of pASARM are active in hypomineralization, we synthesized several ASARM-derived peptide fragments with or without pSer residues (Table 1). Before assessing their biological activities, we tested the stability of the pASARM peptide ID1 at 37 °C up to 5 days. HPLC analysis revealed that ID1 was stable up to 3 days but that undesired byproducts constituted over 10% and 15% of the total peak areas by day 4 and day 5, respectively, compared to stock solutions maintained
Discussion
Studies to understand the effects of ASARM with or without pSer residues on bone and cartilage mineralization and the underlying cellular-biochemical mechanisms have been ongoing for over ten years. As summarized above (see the Introduction), MEPE-ASARM with and without pSer residues inhibit bone mineralization in a variety of cell culture models but with discrepant results reported on whether proliferation and differentiation were also affected (mouse bone marrow stromal cells, Liu et al., 2007
Funding
Yuji Yoshiko was supported in part by the Raffinee International Foundation.
CRediT authorship contribution statement
Tomoko Minamizaki: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Writing - original draft, Writing - review & editing. Kaoru Sakurai: Data curation, Investigation, Visualization. Ikue Hayashi: Data curation, Visualization. Masaaki Toshishige: Data curation, Visualization. Hirotaka Yoshioka: Methodology. Katsuyuki Kozai: Supervision. Yuji Yoshiko: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.
Declaration of competing interest
The authors have declared that no conflict of interest exists.
Acknowledgements
We thank M. Kondo and Y. Sato for their technical assistance. We thank J.E. Aubin, University of Toronto, for valuable discussion and suggestions on our manuscript.
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- 1
Current address, Department of Anatomy, School of Medicine, International University of Health and Welfare, Narita, Japan.
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Tomoko Minamizaki and Kaoru Sakurai contributed equally to this work.