title: Secreted phosphoprotein-24 kDa (Spp24) attenuates BMP-2-stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2 -Macroglobulins From Serum.
abstract: Secreted phosphoprotein-24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W-20-17 mesenchymal stem cells with rhBMP-2, short-term Smad1/5 phosphorylation was inhibited, intermediate-term alkaline phosphatase (ALP) induction was blunted, and long-term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS-PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non-reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween-20 at pH 2.7-8.5. LC-MS/MS analysis of affinity-purified, non-reducing SDS-PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α(2) -macroglobulins (α(2) -macroglobulin [α(2) M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti-thrombin III (Serpin C1), which may protect Spp24 from proteolysis.
authors.affiliation: Geriatric Research, Education and Clinical Center, Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, CA 91343, USA.
authors.forename: Ke-Wei
authors.initials: KW
authors.lastname: Zhao
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Murray
authors.forename: Elsa J Brochmann
authors.initials: EJ
authors.lastname: Murray
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism / Q000494:pharmacology; D001842:Bone and Bones / Q000378:metabolism; D002113:Calcification, Physiologic; D002417:Cattle; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D059748:Proteolysis; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: C525718:BMP2 protein, human; D001798:Blood Proteins; D055396:Bone Morphogenetic Protein 2; D010750:Phosphoproteins; D051785:Smad Proteins; D000511:alpha-Macroglobulins; C091340:secreted phosphoprotein 24; D000990:Antithrombin III; D000469:Alkaline Phosphatase