Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina

A retinol-binding glycoprotein (IRBP) was purified in milligram quantities from the extracellular matrix (interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a K(D) of ~10^-6 M. The holoprotein has λ(max) at 280 nm (E(1 cm)(1%) = 10.99) and at 330 nm (E(1 cm)(1%) = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, α-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of α-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of ~1:3:2. No galactosamine was detected. Observations on the binding of ¹²⁵I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The M(r) of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) and the apparent M(r) was 144,000 IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP, which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with ³H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with ³H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an M(r) reduced by ~5,000.