Paul J. O'Brien

Rhodopsin as a glycoprotein: a possible role for the oligosaccharide in phagocytosis

Abstract Isolated bovine photoreceptor outer segment preparations are capable of transferring both galactose and fucose from the appropriate sugar nucleotides to rhodopsin. Both manganese chloride and Triton, a non-ionic detergent, are required for full activity. A further stimulation of transfer is produced by ATP. No effect is produced by cyclic-AMP, cyclic-GMP, taurine or calcium chloride on either galactose or fucose transfer. The incorporated radio-active sugars co-migrate with rhodopsin on agarose chromatography. Intact retinas incubated in vitro also incorporate galactose into opsin and rhodopsin. Visual pigment labeled with galactose by either method can be purified on agarose columns and hydrolyzed, releasing 94% of the label which co-chromatographs with authentic galactose in two solvent systems. Neither fucose nor galactose is found in rhodopsin but may be added to rhodopsin as a prelude to disc shedding, thereby providing a new recognition site which allows the pigment epithelium to initiate phagocytosis.

Retinal degenerations in the dog III abnormal cyclic nucleotide metabolism in rod-cone dysplasia

In dogs bred to develop rod-cone dysplasia, retinal development is normal until 13 days of age.Afterwards, there is an arrest of visual cell differentiation. Rod inner segments remain diminutive and outer segments fail to elongate as in controls; the outer segments show lamellar disorientation and disorganization. Affected visual cells degenerate, but the degeneration process is more rapid and extensive for rods than cones. Cyclic GMP levels become elevated in affected retinas early during the postnatal differentiation of visual cells; this elevation precedes any morphological evidence of photoreceptor disease. Retinal protein synthesis is normal during the time that retinal cGMP levels are rising above control values. The results indicate that the biochemical abnormality which results in elevated retinal cGMP levels is the earliest recognized defect in rod-cone dysplasia.

The acylation of rat rhodopsin in vitro and in vivo

Rat retinas were incubated with [3H] palmitate and [14C] leucine and subsequently detergent-extracted. Glycoproteins were isolated on Con A-Sepharose columns and separated by gel electrophoresis. Leucine labeled the newly synthesized opsin but palmitate was esterified to both mature rhodopsin and newly synthesized opsin which migrated more slowly because of its untrimmed oligosaccharide chains. Crude rod outer segments were found to contain most of the palmitate-labeled mature rhodopsin, while the retinal debris contained most of the doubly labeled newly synthesized opsin. Homogenization of double-labeled retinas followed by centrifugation in a linear sucrose gradient gave rise to several bands of particles including purified rod outer segments, a Golgi-enriched fraction and a pellet enriched in endoplasmic reticulum. Newly synthesized opsin was first found in the pellet at the earliest incubation times and subsequently appeared in the Golgi fraction and finally in the rod outer segments. Palmitate-labeled mature rhodopsin was found only in the rod outer segments. It appeared at the earliest time points and increased with time. Thus the acylation of opsin occurs in the endoplasmic reticulum, shortly after polypeptide synthesis, and in the rod outer segments, the latter possibly as an exchange reaction. Most of the newly synthesized opsin remained in the pellet and did not pass through the Golgi to the rod outer segments. Intravitreal injection of [3H] palmitate and [14C] leucine gave rise to doubly labeled opsin that appeared to remain untrimmed for at least 6 hr in vivo. After 17 hr, both labels were found only in mature rhodopsin, thus accumulation of new molecules in the endoplasmic reticulum may occur in vivo. In addition, leucine maximally labeled the opsin-rhodopsin pool early in the first day whereas palmitate did not maximally label rhodopsin until 2- or 3 days post injection. Moreover, while leucine label was lost at day 9 because of rod outer-segment renewal and shedding, the palmitate label in rhodopsin remained unchanged. Thus, palmitate labeling in vivo reflects the pattern seen in vitro with a prolonged equilibration of rod outer-segment rhodopsin with the fatty-acid pool, probably mediated by a fatty acyl exchange reaction.

Retinal dystrophy in the RCS rat: in vivo and in vitro studies of phagocytic action of the pigment epithelium on the shed rod outer segments.

Abstract The ability of the pigment epithelium (PE) of dystrophic and control RCS rats to phagocytize rod outer segment (ROS) material was examined in vivo and in vitro. In the in vivo studies of animals with fully developed ROS, the number of phagosomes found in the PE increased between 1 and 2 hr after the onset of light in both groups, although at all times the dystrophic animals demonstrated substantially smaller numbers than the controls. During the postnatal development of ROS, the number of phagosomes found at the daily peak increased steadily in the control rats until adult levels were reached at 35 days. In the dystrophic animals the maximum number was observed by 15 days and showed no further increase with age. Organ culture of contro retina-PE explants caused a burst of ROS shedding and phagocytosis, with the maximum number of phagosomes appearing at 2 hr of incubation. The dystrophic retina-PE explants exhibited a very small peak at 4 hr. Recombinations of dystrophic retina with control PE and control retina with dystrophic PE revealed extensive phagocytosis by the control PE but not by the dystrophic PE. These results confirm the previous observation in chimeric rats that the retina of the RCS dystrophic rat is apparently normal while the PE carries the genetic expression of the defect.

Transfer of N-acetylneuraminic acid to incomplete glycoproteins associated with microsomes

Abstract Rat-liver microsomes incubated with 14C -labeled CMP- N -acetylneuraminic acid (CMP-NANA) transfer N -acetylneuraminic acid (NANA) to an endogenous acceptor. The K m for CMP-NANA is 2·10 −6 M. The reaction stops when acceptor sites are filled (50 μμmoles/g liver). The transfer is reduced by 85% if the rats are treated with puromycin 45 min before removal of the liver, but not after exposure to puromucin in vitro . Transfer to a neuraminidase-treated glycoprotein is not affected by the puromycin injection indicating that the endogenous acceptor is depleted after treatment with the antibiotic. The product of endogenous transfer remains associated with microsomes, from which it can be solubilized by detergents or sonication. Sonic extracts give rise to radioactive precipitin lines with antiserum against rat plasma glycoprotein lacking NANA residues.

Phorbol ester- and light-induced endogenous phosphorylation of rat rod outer-segment proteins

We have previously described the presence of a C-kinase in bovine retinal rod outer segments (ROS) (Kapoor and Chader, 1984). In this study, we have labeled rat retinas with freshly neutralized radiolabeled sodium phosphate (32P or 33P) by intravitreal injection and compared the phosphorylation patterns of ROS proteins induced by light and specific activators of the C-kinase phosphorylation system. Except for light treatment, all procedures were carried out in complete darkness using an infrared image converter. Incubation of 33P-labeled retinas in light for 5 min resulted in the phosphorylation of rhodopsin, 80-, 65-, 47-, 44-, and 15,000 MW proteins of crude ROS. Incubation of 33P-labeled retinas with 0.5 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the phosphorylation of several proteins including those at 80-, 65-, 47-, 44-, and 15,000 MW in crude ROS. ROS prepared in complete darkness did not exhibit any phosphorylation of proteins whereas ROS prepared in red light exhibited variable low phosphorylation of 80-, 47-, 44- and 15,000 MW proteins. 1-oleoyl-2-acetylglycerol (OAG) at 500 micrograms ml-1 caused the phosphorylation of the same proteins as observed with TPA. TPA (0.5-500 microM) and OAG (150-500 micrograms ml-1) did not induce rhodopsin phosphorylation. When purified ROS were prepared from 33P-pre-labeled retinas, the complete darkness control did not exhibit phosphorylation of any proteins. TPA, however, induced the phosphorylation of 80- and 65,000 MW proteins and light induced the phosphorylation of 80-, 65,000 MW proteins as well as opsin monomer and dimer. Affinity chromatography of phosphorylated ROS proteins on con A-Sepharose revealed that TPA does not induce rhodopsin phosphorylation whereas light does. Since light and TPA induced the phosphorylation of 80- and 65,000 MW proteins in ROS, it is possible to suggest at least a partial linkage of light- and C-kinase-mediated effects in situ.

Synthesis of interphotoreceptor retinoid-binding protein (IRBP) by monkey retina in organ culture: Effect of monensin

Whole monkey retinas were incubated in short-term organ culture with either radiolabeled amino acids or glucosamine. Soluble retinal proteins and proteins in the culture medium were analyzed by SDS-poly-acrylamide gel electrophoresis. Fluorography showed that the interphotoreceptor retinoid-binding protein (IRBP), a 146,000 Mr glycoprotein localized in the extracellular matrix, is synthesized by the neural retina and rapidly secreted into the medium. Secretion is blocked by 10-5M monensin. No significant IRBP synthesis was observed in the pigment-epithelium-choroid complex. IRBP is thus the major component synthesized and secreted by the neural retina into the interphotoreceptor space. This, and its affinity for retinoid makes it a prime candidate for an extracellular retinoid transport vehicle.

The synthesis of N-acetyl-α- and N-acetyl-β-D-glucosamine 1-phosphates (2-acetamido-2-deoxy-α- and β-D-glucose 1-phosphates)

Abstract 1. 1. The chemical synthesis of N-acetyl-α- and β- D -glucosamine 1-phosphate is described. Crystalline phosphoric acid was condensed with 2- acetamido -1,3,4,6- tetra -O- acetyl -2- deoxy -α- D -glucose . The O-acetyl groups were removed with LiOH which also precipitated excess phosphoric acid as the insoluble lithium salt. Excess LiOH was removed by conversion to NH4OH with an ion exchange resin and subsequent evaporation. 2. 2. The α and β anomers were separated with an ion exchange resin and crystallized: the α anomer as the monohydrate of the dipotassium salt, and the β anomer as the anhydrous disodium salt. 3. 3. The α anomer was active as a substrate for the enzyme UTP: 2-acetylamino-2-deoxy- D -glucose-1-phosphate uridylyltransferase; the β anomer was inactive. 4. 4. The synthesis of glycosyl-β-1-phosphates by this method has not been reported previously.

A quantitative system for studying phagocytosis in pigment epithelium tissue culture

Abstract A quantitative assay for phagocytosis has been described using a heterologous system of chick pigment epithelial culture and radiolabeled bovine rod outer segment suspensions. Electron microscopy was used to verify results obtained in radiolabeled uptake studies. Freezing and mild shearing of the outer segment suspensions do not significantly inhibit phagocytosis. Outer segments radiolabeled with [ 3 H]choline and [ 14 C]mannose showed similar kinetics of phagocytosis. Maximum uptake was between 1 and 2 hr of incubation and approached 1% of the total applied radioactivity for mannose and 2% for choline.

The biosynthesis of rhodopsin in vitro

Abstract Radioactive glucosamine and leucine are incorporated into the outer segments of the rod cells of bovine retinas incubated in vitro. One component of the outer segment labeled in this process is rhodopsin which can be extracted with detergent and purified by sequential chromatography on calcium phosphate-Celite and agarose. Both glucosamine and leucine appear in the rhodopsin of the outer segments after pronounced lags, longer for leucine, shorter for glucosamine. The difference corresponds to the additional time required for newly-synthesized polypeptide to be accumulated at some point in the photoreceptor cell where glycosylation presumably occurs. The lag in glucosamine labeling corresponds to the time required for the fully glycosylated polypeptide to complete the process of assembly of light-sensitive outer segment disc membranes. The overall process agrees with in vivo studies of outer segment renewal. Two other proteins are labeled along with rhodopsin. They can be isolated by the same chromatographic procedures from outer segment extracts. One is labeled and appears in the outer segments prior to rhodopsin and may be the diffusely distributed labeled protein seen in autoradiographic studies. The other is like opsin in that it can bind a chromophore to produce a visual pigment and may be designated as a pre-rhodopsin.