03585nas a2200589 4500008004100000022001400041245010200055210006900157260001500226300001400241490000800255520182400263653001202087653001802099653002902117653001302146653003102159653002202190653002802212653004602240653002902286653002702315653001302342653002002355653001702375653001102392653003302403653002202436653001502458653000902473653002102482653001302503653001402516653002002530653004302550653002402593653001702617653003002634653004102664653001402705653002302719100001702742700002602759700002002785700002102805700002602826700001802852700001802870700002702888700002002915856006002935 2015 eng d a1524-457100aFunctional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.0 aFunctional Dissection of the CCBE1 Protein A Crucial Requirement c2015 May 8 a1660-16690 v1163 a
RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.
OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 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, although 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, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome 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.
10aAnimals10aBinding Sites10aCalcium-Binding Proteins10aCollagen10aCraniofacial Abnormalities10aEndothelial Cells10aEpidermal Growth Factor10aGene Expression Regulation, Developmental10aGene Knock-In Techniques10aGenital Diseases, Male10aGenotype10aGestational Age10aHEK293 Cells10aHumans10aLymphangiectasis, Intestinal10aLymphatic Vessels10alymphedema10aMice10aMice, Transgenic10aMutation10aPhenotype10aProtein Binding10aProtein Interaction Domains and Motifs10aSignal Transduction10aTransfection10aTumor Suppressor Proteins10aVascular Endothelial Growth Factor C10aZebrafish10aZebrafish Proteins1 aRoukens, Guy1 aPeterson-Maduro, Josi1 aPadberg, Yvonne1 aJeltsch, Michael1 aLeppänen, Veli-Matti1 aBos, Frank, L1 aAlitalo, Kari1 aSchulte-Merker, Stefan1 aSchulte, Dörte uhttp://circres.ahajournals.org/content/116/10/1660.long