Retinoschisin forms a multimolecular complex with extracellularmatrix and cytoplasmic proteins: interactions with β2 laminin andαB-crystallin

[摘要] Purpose: X-linked juvenile retinoschisis is a rare early-onsetretinal degeneration characterized by the formation of cysts and loss ofthe electroretinogram "b" wave. The affected gene normally codes forretinoschisin (Rs1), a secreted protein containing a large discoidinhomology domain. Rs1 seems to be principally synthesized in thephotoreceptors, but its structure and spectrum of effects when mutatedindicates association with other proteins. The present study searchedfor retinal proteins capable of interacting with Rs1.Methods: Western blotting and RT-PCR of isolated outer nuclear(photoreceptors), inner nuclear and ganglion cell layers, and cellculture compartments were performed to verify sites of Rs1 synthesis anddistribution. Potential Rs1 binding partners were searched for withaffinity columns generated using specific Rs1- and β2laminin-antisera. Following loading with total protein extracts fromporcine retina, bound proteins were acid eluted and visualized byCoomassie blue staining of SDS-polyacrylamide gels, and selected bandswere excised for tryptic peptide digestion and sequencing. Using singleand double labeled immunohistochemistry, candidate binding partnerdistributions with that of Rs1.Results: Whereas Rs1 mRNA was confined to the outer nuclear layer,Rs1 protein was found throughout the retina, including within theganglion cell layer. One protein that was retained on Rs1 affinitycolumns was identified as αB crystallin, which showed partiallyoverlapping distribution with Rs1 in the retina, mainly in theinterphotoreceptor matrix and outer plexiform layer. Also, β2laminin columns retained Rs1, and again shared partial distributionpatterns. Finally, unidentified peanut agglutinin-binding proteins fromthe retina also bound to Rs1, αB crystallin and β2 laminin.Conclusions: Taken together, these data demonstrate that Rs1associates with different proteins during its synthesis and secretion,forming a multimolecular complex which presumably forms a stabilizingscaffold for retinal synapses, and possibly overall tissue integrity.

[学科分类] 生物化学/生物物理