Abstract
Purpose: :
Retinoschisin is a putative signal protein of the retina. Mutations in its gene cause X–linked retinoschisis (XLRS), a juvenile macular degeneration manifesting compromised retinal integrity. This phenotype, together with functional domain analysis of retinoschisin suggest that this protein is potentially involved in adhesion. It could act as an adhesion protein by itself or could interact with extracellular adhesion proteins. Since collagen is an important component of cell–matrix adhesion, we tested if retinoschisin interacts with collagen and contribute to adhesion in the retina.
Methods: :
We first generated recombinant retinoschisin (rRS). rRS was incubated with immobilized collagen. After washes, elution fractions were collected and subjected to SDS–PAGE and Western blotting. We also used a substrate adhesion assay to examine if retinoschisin is involved in adhesion and if interaction with collagen affects adhesion. Bovine serum albumin (BSA), collagen and rRS were used as substrates. BSA and collagen were applied alone or after mixing with rRS. Adhesion was evaluated by estimating the number of dissociated retinal cells that remained on the substrate after extensive washing.
Results: :
1) Monomeric retinoschisin was eluted from the column packed with immobilized collagen. 2) The substrate adhesion assay showed that more cells were retained by collagen and rRS than BSA. The number of cells adhered to the mixture of rRS and collagen was significantly fewer than that to either of the proteins alone.
Conclusions: :
Our pull–down assay indicates a direct interaction of retinoschisin with collagen. This interaction has a significant functional consequence. Retinoschisin and collagen alone act as adhesion protein to retinal cells, but their interaction changes their adhesive properties. These results provide insights into the molecular basis of XLRS.
Keywords: retinal degenerations: cell biology • photoreceptors • retinal adhesion