A Carboxy Terminal BMP/TGF-b Binding Site in Secreted Phosphoprotein 24 kD Independently Affects BMP-2 Activity Haijun Tian, 1,2 * Chen-Shuang Li, 3 Ke-Wei Zhao, 4 Jeffrey C. Wang, 2 M. Eugenia L. Duarte, 5 Cynthia L. David, 6 Kevin Phan, 2 Elisa Atti, 7 Elsa J. Brochmann, 4,8,9 and Samuel S. Murray 4,8,9 1 Department of Orthopaedic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China 2 Department of Orthopaedic Surgery, University of California, Los Angeles, California 90024 3 Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing 100081, P.R. China 4 Research Service, VA Greater Los Angeles Healthcare System, North Hills, California 91343 5 National Institute of Traumatology and Orthopaedics, Federal University of Rio de Janeiro, Rio de Janeiro 21941, Brazil 6 Center for Toxicology, University of Arizona, Tucson, Arizon 85721 7 School of Dentistry, University of California, Los Angeles, California 90024 8 Geriatric Research, Education and Clinical Center, VA Greater Los Angeles Healthcare System, North Hills, California 91343 9 Department of Medicine, University of California, Los Angeles, California 90024 ABSTRACT Secreted phosphoprotein 24 kD (spp24) is a bone matrix protein isolated during attempts to identify osteogenic proteins. It is not osteogenic but performs other important roles in the regulation of bone metabolism, at least in part, by binding to and affecting the activity of members of the BMP/TGF-b family of cytokines. Spp24 exists in a number of forms that preserve the N-terminus and are truncated at the C-terminus. The hypothesized cytokine binding domain is present within the cystatin domain which is preserved in all of the N-terminal products. In this report, we describe a C-terminal fragment that is distinct from the cystatin domain and which independently binds to BMP-2 and TGF-b. This fragment inhibited BMP-2 activity in an ectopic bone forming assay. A shorter C-terminal product did not inhibit BMP-2 activity but improved bone quality induced by BMP-2 and produced increased calcium deposition outside of bone. Spp24 has been used to develop several potential therapeutic proteins. These results provide more information on the function of spp24 and provide other materials that can be exploited for clinical interventions. J. Cell. Biochem. 116: 667–676, 2015. © 2014 Wiley Periodicals, Inc. KEY WORDS: SECRETED PHOSPHOPROTEIN 24 kD; BONE MORPHOGENETIC PROTEIN 2; BONE RESEARCH S ecreted phosphoprotein 24 kD (spp24; UniProtKB accession number for bovine spp24: Q27967; UniProtKB accession number for human spp24: Q13103) is a bone matrix protein the function of which is only now being elucidated. While investigators working several decades ago isolated this protein, or fragments of this protein, in extracts of bone matrix proteins that were described Grant sponsor: Research Service of the Department of Veterans Affairs; Grant number: 1I01BX000511; Grant sponsor: NIEHS; Grant number: ES06694; Grant sponsor: NIH/NCI; Grant number: CA023074. *Correspondence to: Haijun Tian, MD, PhD, 415 Fengyang Rd, Department of Orthopaedic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, 200003, P.R. China. E-mail: haijuntianmd@gmail.com Manuscript Received: 21 January 2014; Manuscript Accepted: 19 November 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 22 November 2014 DOI 10.1002/jcb.25023  © 2014 Wiley Periodicals, Inc. 667 ARTICLE Journal of Cellular Biochemistry 116:667–676 (2015)