Retinoschisin forms a multi-molecular complex with extracellular matrix and cytoplasmic proteins: interactions with...

X-linked juvenile retinoschisis is a rare early-onset retinal degeneration characterized by the formation of cysts and loss of the electroretinogram "b" wave. The affected gene normally codes for retinoschisin (Rs1), a secreted protein containing a large discoidin homology domain. Rs1 seems to be principally synthesized in the photoreceptors, but its structure and spectrum of effects when mutated indicates association with other proteins. The present study searched for retinal proteins capable of interacting with Rs1.

Western blotting and RT-PCR of isolated outer nuclear (photoreceptors), inner nuclear and ganglion cell layers, and cell culture compartments were performed to verify sites of Rs1 synthesis and distribution. Potential Rs1 binding partners were searched for with affinity columns generated using specific Rs1- and beta2 laminin-antisera. Following loading with total protein extracts from porcine retina, bound proteins were acid eluted and visualized by Coomassie blue staining of SDS-polyacrylamide gels, and selected bands were excised for tryptic peptide digestion and sequencing. Using single and double labeled immunohistochemistry, candidate binding partner distributions with that of Rs1.

Whereas Rs1 mRNA was confined to the outer nuclear layer, Rs1 protein was found throughout the retina, including within the ganglion cell layer. One protein that was retained on Rs1 affinity columns was identified as alphaB crystallin, which showed partially overlapping distribution with Rs1 in the retina, mainly in the interphotoreceptor matrix and outer plexiform layer. Also, beta2 laminin columns retained Rs1, and again shared partial distribution patterns. Finally, unidentified peanut agglutinin-binding proteins from the retina also bound to Rs1, alphaB crystallin and beta2 laminin.

Taken together, these data demonstrate that Rs1 associates with different proteins during its synthesis and secretion, forming a multimolecular complex which presumably forms a stabilizing scaffold for retinal synapses, and possibly overall tissue integrity.