Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

Site-specific cleavage of BMP4 by furin, PC6, and PC7.

Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin- and PC6-selective inhibitor alpha(1)-PDX. By contrast, alpha(1)-PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to alpha(1)-PDX. Consistent with this prediction, an alpha(1)-PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion.

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain.

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.