Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

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Abstract
The interphotoreceptor retinoid-binding protein (IRBP) was isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC) and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a MW of 106,000 .+-. 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 .+-. 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBP have isoelectric points between 6.0 and 7.0. The fluorescence emission .lambda.max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation .lambda.max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids accounts for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated protiens. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBP in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.