BMP-1 disrupts cell adhesion and enhances TGF-ß activation through cleavage of the matricellular protein thrombospondin-1.

Bone morphogenetic protein 1 (BMP-1) is an important metalloproteinase that synchronizes growth factor activation with extracellular matrix assembly during morphogenesis and tissue repair. The mechanisms by which BMP-1 exerts these effects are highly context dependent. Because BMP-1 overexpression induces marked phenotypic changes in two human cell lines (HT1080 and 293-EBNA cells), we investigated how BMP-1 simultaneously affects cell-matrix interactions and growth factor activity in these cells. Increasing BMP-1 led to a loss of cell adhesion that depended on the matricellular glycoprotein thrombospondin-1 (TSP-1). BMP-1 cleaved TSP-1 between the VWFC/procollagen-like domain and the type 1 repeats that mediate several key TSP-1 functions. This cleavage induced the release of TSP-1 C-terminal domains from the extracellular matrix and abolished its previously described multisite cooperative interactions with heparan sulfate proteoglycans and CD36 on HT1080 cells. In addition, BMP-1-dependent proteolysis potentiated the TSP-1-mediated activation of latent transforming growth factor-ß (TGF-ß), leading to increased signaling through the canonical SMAD pathway. In primary human corneal stromal cells (keratocytes), endogenous BMP-1 cleaved TSP-1, and the addition of exogenous BMP-1 enhanced cleavage, but this had no substantial effect on cell adhesion. Instead, processed TSP-1 promoted the differentiation of keratocytes into myofibroblasts and stimulated production of the myofibroblast marker α-SMA, consistent with the presence of processed TSP-1 in human corneal scars. Our results indicate that BMP-1 can both trigger the disruption of cell adhesion and stimulate TGF-ß signaling in TSP-1-rich microenvironments, which has important potential consequences for wound healing and tumor progression.

Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain.

UNLABELLED: Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation. SIGNIFICANCE: An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications.

Proteolytic control of TGF-ß co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics.

The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-ß superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-ß co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-ß was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-ß co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .