Abstract
Purpose :
Transport of vitamin A/retinol/ROL to peripheral tissues including the eye via the circulation is mediated by plasma retinol-binding protein (RBP4). STRA6, the only known membrane receptor for cellular RBP4-ROL is expressed in the eye, but not in systemic tissues involved in dietary vitamin A uptake and storage. Here, we investigated our hypothesis that the recently identified retinol binding protein receptor 2 (Rbpr2) has high affinity binding for RBP4 and that this physical interaction is critical for ROL transport to the eye during development.
Methods :
Sequence alignment was performed to map previously proposed RBP4 binding residues on Rbpr2. Site-directed mutagenesis was used to alter these sequences in zebrafish rbpr2 gene. Membrane localization patterns of wild-type Rbpr2 and Rbpr2-mutants targeting the RBP4 binding residues was assessed using immunohistochemistry and confocal microscopy in NIH3T3 cells. Vitamin A uptake studies were performed using [3H]retinol-RBP4 in cultured cells. Co-immunoprecipitations assays were performed to access ability of Rbpr2-RBP4 mutants to bind circulatory RBP4. CRISPR/Cas9 genome editing technology was used to generate zebrafish mutants targeting the RBP4-ROL binding domain. HPLC analysis was used to quantify ocular retinoids.
Results :
Modeling and docking studies confirmed conserved RBP4 binding residues in zebrafish Rbpr2. Immunohistochemical and confocal analysis revealed that Rbpr2 mutants affecting the RBP4 binding sites had normal membrane expression patterns but reduced vitamin A uptake capabilities. Co-immunoprecipitations assays showed significantly decreased RBP4 binding by Rbpr2-mutants. Zebrafish rbpr2-mutants affecting the RBP4 binding domains, showed smaller eyes with largely intact retinal lamination. Immunofluorescence analysis showed early loss of rod, followed by progressive loss of cone photoreceptor proteins, associated with suboptimal ocular retinoids.
Conclusions :
The studies reported here demonstrate that the second vitamin A transporter Rbpr2 in zebrafish, like STRA6, contains a functional RBP4 binding domain that is critical for vitamin A delivery to the vertebrate eye during development. Future studies utilizing a CRISPR mouse model targeting the individual RBP4 binding residues in RBPR2 is proposed.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.