Abstract
Purpose: :
A missense mutation in the CTRP5 gene is implicated in a complex ocular phenotype of early–onset long anterior lens zonules and late–onset retinal degeneration (LORD). CTRP5 is co–transcribed with MFRP as bicistronic gene. Null mutation in MFRP is associated with nanophthalmos in human and a splice site mutation causes retinal degeneration in mouse (rd6). Studies are carried out to understand the role of CTRP5 and MFRP genes in ocular physiology.
Methods: :
Localization of CTRP in MDCK cells, adult albino mouse and human eye sections was determined by immunofluorescence and immuno electron microscopy using CTRP5 and marker protein specific antibodies. Interaction of CTRP5 and MFRP proteins in human and mouse ocular tissues and also in COS–7 cells co–transfected with expression constructs of V5–tagged CTRP5 and Xpress–tagged MFRP was determined by immunoprecipitation (IP) and immunoblotting (IB).
Results: :
CTRP5 is localized to the RPE membrane in hexagonal punctate fashion and co–localized with ZO–1 and F–actin as beads on a string. CTRP5 also co–localizes with Connexin 43 and Ezrin on the RPE apical processes. In the ciliary epithelial tissue, CTRP5 shows partial co–localization with F–actin and complete co–localization with Connexin 43. In MDCK cells endogenous CTRP5 protein is localized to the cell junctions and apical membranes. In these cells CTRP5 is co–localized with ZO–1 at the cell junctions and with MFRP on the apical membranes. Co–immunoprecipitaion analysis of protein extracts from human RPE–choroid, mouse eye and co–transfected COS–7 cells with CTRP5, MFRP or tag specific antibodies suggests putative interaction between CTRP5 and MFRP proteins.
Conclusions: :
CTRP5 is localized to the cellular junctions as well as the apical membrane of polarized epithelial cells, while MFRP is detected only on the apical membrane. In addition, CTRP5 and MFRP are found to interact with each other in tissues and cultured cells co–expressing these proteins, indicating that this set of ocular bicistronic genes are likely to be functionally related and participate in a common cellular pathway.
Keywords: proteins encoded by disease genes • retinal degenerations: cell biology • retinal pigment epithelium