In vivo biosynthesis of cholesterol in the rat retina.

Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of [3H]acetate. HPLC analysis of retinal non-saponifiable lipid extracts revealed co-elution of radioactivity with endogenous cholesterol mass within 4.5 h post-injection. Incorporation of [3H]acetate into cholesterol was markedly reduced by co-injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB-598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo.

Isoprenoid lipid metabolism in the retina: dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes.

Frogs were injected intravitreally with [3H]acetate, and the formation of [3H]-labeled squalene and cholesterol in the retina and their incorporation into rod outer segment (ROS) membranes were evaluated biochemically over a 60-day time course. ROS [3H]squalene specific activity was maximal by 1-3 days, then declined with a half-time of approximately 20-30 days. In contrast, the specific activity of ROS [3H]cholesterol initially increased to a level substantially less than that of [3H]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS cholesterol. When [3H]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% of those recovered from the ipsilateral retina; hence, systemic contributions to de novo synthesis were obviated. Long-term (> or = 8 hr) in vitro incubations of isolated retinas with [3H]acetate resulted in incorporation of [3H]-labeled sterols and squalene into ROS, at levels comparable to those observed in ROS from companion incubated eyecup preparations and from retinas 8 hr after intravitreal injection of [3H]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with respect to isoprenoid lipid metabolism, and suggest that de novo synthesis within the neural retina is responsible for generating most, if not all, of the [3H]squalene and [3H]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy poisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited marked suppression of incorporation of newly synthesized protein into the ROS, while [3H]squalene incorporation was only minimally reduced, compared with frogs maintained at 22 degrees C. These results are consistent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane proteins.

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid.

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.