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.

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

“…This domain also binds transforming growth factor-␤ (TGF-␤) resulting in increased apoptosis (55). SPP24 has also been observed by us and others (65) to exist in a high mass complex bound to ␣-2-macroglobulin and other proteins in serum (supplemental Table S3). SPP24 is present in smooth muscle, epithelial cells and is released from macrophages and T-cells in response to heightened levels of TNF-␣.…”

Fibroblastic Synoviocytes Secrete Plasma Proteins Via α2‐Macroglobulins Serving as Intracellular and Extracellular Chaperones

“…Induction of experimental arthritis increases the expression of Spp24 in joint cartilage by more than 30‐fold, an increase that is greater than that of any other protein [Meng et al, ]. We reported that Spp24 is present in serum in a high molecular weight (>500 kDa) disulfide‐bonded complex with A2Ms (A2M itself, PZP, and C3) and A1AT [Zhao et al, ]. In studies aimed at characterizing the SF proteome of patients with RA and OA, Spp24 was found to be present if A2M, its abundant carrier protein, had not first been depleted to enhance the detection of less abundant proteins [Balakrishnan et al, ].…”

HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface

“…A small fraction of these proteins (∼10–20% in most cases) can also be detected in the basolateral side (bottom well). In addition, all plasma proteins in the secretion media resembled that of A2M/C3 (360–720 kDa, Zhao et al, []) in spite of their differing molecular weights. These results suggested that all the proteins tested exist as a high molecular weight form, most likely with C3 which serves as an intracellular and extracellular chaperone as A2M also seen in synoviocytes [Zhao et al, ].…”