Identification of the growth factor–binding sequence in the extracellular matrix protein MAGP-1

Abstract

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor–binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1–TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid–long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.

Molecular structure and function of microfibrillar‐associated proteins in skeletal and metabolic disorders and cancers

“…Together, these findings indicate that TGF‐β mediated by MFAP2 has protective effects on metabolic stress, and lack of TGF‐β could result in metabolic dysfunction in individuals (Craft et al, 2014). In line with this, the biological analysis showed that MFAP2 was able to bind to TGF‐β and BMP via its highly acidic sequence near the N terminus (Broekelmann, Bodmer, & Mecham, 2020). In this study, MFAP2 was found to specifically bind to active TGF‐β1, but not to latent TGF‐β1, and in the absence of MFAP2, active TGF‐β1 did not bind fibrillin, indicating that MFAP2 plays an active role in TGF‐β signaling in the ECM (Broekelmann et al, 2020).…”

“…In line with this, the biological analysis showed that MFAP2 was able to bind to TGF‐β and BMP via its highly acidic sequence near the N terminus (Broekelmann, Bodmer, & Mecham, 2020). In this study, MFAP2 was found to specifically bind to active TGF‐β1, but not to latent TGF‐β1, and in the absence of MFAP2, active TGF‐β1 did not bind fibrillin, indicating that MFAP2 plays an active role in TGF‐β signaling in the ECM (Broekelmann et al, 2020). Further, increased gonadal fat pad mass and hyperglycemia were also present in Mfap2(−/−) mice at the young age of 2 months, and peaked by 6 months (Walji et al, 2016), indicating a role of MFAP2 in fat metabolism in an age‐dependent manner.…”

“…plays an active role in TGF-β signaling in the ECM (Broekelmann et al, 2020). Further, increased gonadal fat pad mass and hyperglycemia were also present in Mfap2(−/−) mice at the young age of 2 months, and peaked by 6 months (Walji et al, 2016), indicating a role of MFAP2 in fat metabolism in an age-dependent manner.…”

Rab31-dependent regulation of transforming growth factor ß expression in breast cancer cells

“…Since neither ELISA nor Western blot analyses using antibodies directed to mature TGF-ß were sensitive enough to detect active TGF-ß1, we used a highly sensitive, cell-based bioassay (Fig. 7 A) that allows detection of human TGF-ß levels as low as 1 pg/ml versus 31.2 pg/ml by the ELISA approach (Beaufort et al 2014 ; Broekelmann et al 2020 ; Johnston et al 2017 ; Tesseur et al 2006 ). Concentrated supernatants from CAMA-1 Rab31 and vector transfectants were left untreated to measure active TGF-ß levels or heated to 65 °C to activate the pool of latent TGF-β in order to allow the determination of the total TGF-ß content.…”

“…To measure active TGF-ß, embryonic fibroblasts derived from TGF-ß-deficient mice, and stably transfected with an expression plasmid encoding for the secreted alkaline phosphatase (SEAP) under the control of a Smad2-responsive promoter were used (MFB-F11 cells; [Beaufort et al 2014 ; Broekelmann et al 2020 ; Johnston et al 2017 ; Tesseur et al 2006 ). A density of 2 × 10 4 MFB-F11 cells per well were seeded in 96-well plates (Corning Inc., Corning, NY) and grown for 24 h at 37 °C.…”