Proteoglycans in the mouse interphotoreceptor matrix. VI. Evidence for photoreceptor synthesis of chondroitin sulfate proteoglycan using genetically fractionated retinas

Abstract

To determine the role of photoreceptors in the synthesis of chondroitin sulfate proteoglycan (CS-PG) present in the interphotoreceptor matrix (IPM), 35 SO 4 2− was used as a tracer for comparison of proteoglycans synthesized in vitro in the absence of the pigment epithelium by normal retinas and retinas from retinal degeneration ( rd) mice at stages before and after photoreceptor degeneration. Isolated retinas from 10 day post-partum (P-10) pups, adult normal mice (C57BL/6J ++/++) and retinal degeneration mice (C57BL/6J rdle/rdle) were incubated for 7 hr with 35 SO 4 2− to label newly synthesized sulfated proteoglycans. At P-10, rd retinas have not undergone extensive photoreceptor degeneration, whereas in the adult retinas from this strain, only a few cone photoreceptors remain. At the termination of the labeling period, proteoglycans in the incubation medium and those remaining in guanidine hydrochloride (GuHCl) extracts of the retina were analysed separately and identified by their susceptibility to enzymatic or nitrous acid depolymerization. At P-10, no significant differences were observed in the types or sizes of newly synthesized proteoglycan in normal and rd retinas. Medium samples from P-10 retinas contained near equal amounts of 35S-labeled CS-PG and heparan sulfate proteoglycan (HS-PG), while in GuHCl extracts, approximately 90% of the 35 SO 4 2− was incorporated into HS-PG, with the remainder found in CS-PG. Comparisons of adult tissue revealed a divergence of proteoglycan synthesis profiles. Retinas from normal adults label predominately CS-PG. [ 35S]proteoglycan from normal retina incubation medium was approximately 96% CS-PG, and GuHCl extracts were about 73% CS-PG. From adult rd retinas these values were 18 and 10%, respectively. Per retina, this shows the rd retinas labeling less than 4% of the medium CS-PG, and about 50% of the GuHCl extractable CS-PG compared to normal retinas. Labeled HS-PG comprised about 28% of the normal retina GuHCl extracts, but was not detected in the incubation medium. In contrast, HS-PG synthesis accounted for about 76% of the medium proteoglycan label, and about 85% of the extracted proteoglycan in the adult rd retina. In fact, 35 SO 4 2− labeling of HS-PG in the rd retina GuHCl extracts exceeded by 1000% the level observed in normal retina extracts on a per retina basis. Retinas from both strains incorporate significant amounts of 35 SO 4 2− into proteins with rd achieving higher specific activity. IRBP was identified as a 35 SO 4 2− labeled protein by immunoadsorption from aliquots of the incubation medium. In P-10 normal and rd retinas newly synthesized IRBP was readily detected. The adult rd retinas synthesized only 4–9% of the amount synthesized by the normal adult retina. We found that the adult rd retinas secrete minimal amounts of CS-PG and IRBP, correlating with the paucity of surviving photoreceptors. Collectively these results indicate that the retinal contribution to IPM CS-PG is predominantly an activity of the photoreceptors.