XB-ART-13244Exp Eye Res. April 1, 1999; 68 (4): 399-410.
Interphotoreceptor retinoid-binding protein (IRBP) is rapidly cleared from the Xenopus interphotoreceptor matrix.
The interphotoreceptor matrix (IPM) is a highly-organized extracellular matrix critical to retinal development and function. Although the concentrations of its components are carefully regulated, little is known about the mechanisms of this regulation. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant soluble protein component of the IPM. Although its rate of clearance is thought to be an important factor regulating the concentration of IRBP within the IPM, no study has measured the rate of its extracellular turnover. Here we determine the rate of turnover of matrix IRBP in Xenopus. The rate of IRBP turnover was estimated by measuring the loss of radioactivity from protein labeled by a single injection of a radiolabeled protein precursor. To provide an estimate of the rate of IRBP turnover, we have examined the following issues: (1) Quantitative extraction of IRBP from the IPM for biochemical analysis. (2) Routes of delivery of radiolabeled precursor to achieve a pulse label in vivo. (3) Selection of labeled precursor in order to minimize reutilization of radiolabel. Using Western blot analysis, immunoprecipitation and immuno-electron microscopy, we found that IRBP can be quantitatively extracted from the IPM by a simple saline wash. IRBP was radiolabeled by systemic or intravitreal injection of either [35S]methionine or carboxyl-terminal labeled [1-14C]leucine. The specific activity of matrix IRBP was determined by either phosphorimaging or fluorography of Coomassie blue-stained SDS-polyacrylamide gels. Intravitreal injection of tracer was more effective than systemic delivery in achieving a pulse of radiolabel to the retina. This may be due to intravitreal injection allowing the body to act as a ''sink'' for radiolabeled amino acid. When radiolabeled precursor was delivered by intravitreal injection, the calculated half-life of matrix IRBP using [35S]methionine was 25. 6+/-0.82 hr; in contrast, it was 10.7+/-2.9 hr using [1-14C]leucine. The faster apparent IRBP turnover using [1-14C]leucine is interpreted in context of the early decarboxylation of leucine during its degradation. Our results demonstrate rapid turnover of IRBP in the Xenopus IPM in vivo and suggest that the IPM is a dynamic structure undergoing continuous renewal.
PubMed ID: 10192797
Article link: Exp Eye Res.
Grant support: EY09412 NEI NIH HHS
Genes referenced: grb10 myh3 rbp3
Antibodies referenced: Rbp3 Ab1
Article Images: [+] show captions
|Fig. 1. Metabolic labeling of Xenopus IRBP and opsin by intravitreal injection of [$&S]methionine. (A) Soluble fraction of the interphotoreceptor matrix (SDS-8%PAGE). (B) Retinal membranes (SDS-8%PAGE). The gels were stained with Coomassie blue (Protein) and exposed to X-ray film for C3 days (Fluorogram). Arrowhead, IRBP (Mr¯124); bracket, opsin.|
|Fig. 2. Identi®cation of IRBP by immunoprecipitation. Ocular proteins were metabolically labeled by intravitreal injection of [$&S]methionine. Twenty-seven hours following the injection, soluble fractions of the retina, interphotoreceptor matrix and RPE-eyecup were subjected to SDS-10% PAGE (lane A in each panel). IRBP (arrow) was immunoprecipitated using anti-Xenopus IRBP serum (lane B in each panel). Fluorograms were exposed for C4 days.|
|Fig. 3. Immuno-electron micrograph of the apical RPE from control (undetached) retina incubated with anti-Xenopus IRBP serum followed by protein A-labeled 10 nm colloidal gold. The insert (lower power, provided for orientation) shows numerous microvilli between the apical RPE and an outer segment tip (OS). At higher magni®cation, extensive immunogold labeling (arrowheads) is visible between the microvilli (asterisks). Sections treated with pre-immune serum showed no speci®c labeling (data not illustrated).|
|Fig. 4. Immuno-electron micrographs of a saline-washed RPE-eyecup. Light-adapted adult Xenopus were killed at midmorning, and detached RPE-eyecups were washed with saline. Sections were incubated with anti-Xenopus IRBP serum followed by protein A-labeled 10 nm colloidal gold. In contrast to the unwashed retina (Fig. 3), the extracellular region (asterisks) is devoid of gold label. IRBP was not detected coating the extracellular surface of the RPE or its microvilli (arrowheads). (A) and (B) IRBP contained within vesicles (v) in the apical RPE cytoplasm. These structures are shown at higher magni®cation in (C) and (D). The gold label is often localized to the inner surface of the vesicle (open arrows). Sections treated with pre-immune serum showed no speci®c labeling (data not illustrated). Bar¯0±5 lm in (A) and 1±0 lm in (B), (C), (D).|
|Fig. 5. Specific activities of IRBP and opsins following systemic injection of [$&S]methionine. Juvenile Xenopus were injected in the dorsal lymph sac with 500 lCi of [$&S]methionine. The speci®c activities of IRBP and opsin were measured at various timepoints following injection. (A) Representative Coomassie stained SDS polyacrylamide gels of the soluble matrix (Matrix, arrow indicates IRBP) and the retinal membrane fraction (Membranes, bracket indicates opsin). (B) Representative PhosphorImager bands ($&S) for both IRBP (arrow) and opsin (bracket) as well as the corresponding Coomassie blue-stained band (CB) from the same eye. Time (days post-injection) is indicated at the top of the panel. (C) Specific activities of IRBP and opsin as a function of time following injection. IRBP speci®c activity (E) remains constant, while opsin specific activity (+) increases over time.|
|Fig. 6. Turnover of IRBP and opsin following intravitreal injection of [$&S]methionine. (A) Representative ¯uorograms ($&S) and Coomassie-blue stained gels (CB) are shown for both IRBP and opsin. (B) IRBP (E) and opsin (D) speci®c activities as a function of time following injection. IRBP in the IPM has a calculated half-life of 25³0±82 hr.|
|Fig. 7. Turnover of IRBP and opsin following intravitreal injection of [1-"%C]leucine. (A) Representative ¯uorograms ("%C) and Coomassie-blue stained gels (CB) are shown for both IRBP and opsin. (B) IRBP (E) and opsin (D) speci®c activities as a function of time following injection. IRBP in the IPM has a calculated half-life of 10±5³2±9 hr.|