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J. Biol. Chem., Vol. 282, Issue 36, 26002-26013, September 7, 2007

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Association of Specific Proteolytic Processing of Bone Sialoprotein and Bone Acidic Glycoprotein-75 with Mineralization within Biomineralization Foci^*

Nichole T. Huffman, J. Andrew Keightley, Cui Chaoying^¶, Ronald J. Midura^||, Dinah Lovitch, Patricia A. Veno, Sarah L. Dallas, and Jeff P. Gorski¹

From the Bone Biology Program, Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, Missouri 64108, Biological Mass Spectrometry and Proteomics Facility, Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110, ^¶Tibet University Medical College, Lhasa 850002, Tibet, China, and the ^||Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15–25-µm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45–50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.

Received for publication, February 15, 2007 , and in revised form, June 19, 2007.

^* This work was supported by National Institutes of Health Grants R21 DE14619 (to J. P. G.) and R01 AR052775 (to J. P. G.) and small grants from the Women's Council of the University of Missouri, Kansas City (to N. T. H.). Parts of this research were presented in preliminary form at the annual meeting of the American Society for Biochemistry and Molecular Biology, June 12–16, 2004, Boston, MA; the American Society for Bone and Mineral Research, September 23–27, 2005, Nashville, TN; and the American Society for Bone and Mineral Research, September 15–19, 2006, Philadelphia, PA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Bone Biology Program, Oral Biology, School of Dentistry, 625 East 25th St., University of Missouri, Kansas City, MO 64108. Tel.: 816-235-2537; Fax: 816-235-5524; E-mail: Gorskij{at}umkc.edu.

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