Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain
Rationale: CCBE1 is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam Syndrome (HS). Recently, CCBE1 has emerged as a crucial regulator of VEGFC signaling.
Objective: CCBE1 is a secreted protein characterized by two EGF domains and two collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro.
Methods and Results: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, while CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1∆EGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a HS mutation within the collagen domain has a stronger effect than a HS mutation within the EGF domain.
Conclusions: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.
- Hennekam Syndrome
- vascular endothelial function
- vascular endothelium
- vascular endothelial growth factor
- Received July 31, 2014.
- Revision received March 25, 2015.
- Accepted March 26, 2015.