Barbara J. McLaughlin

Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina

The pigmented epithelium of the vertebrate retina phagocytizes the discarded tips of photoreceptors and it is likely that a specific cellular recognition process is involved in this phenomenon. The apical surface of retinal pigmented epithelium (RPE) contains microvilli which interdigitate with the outer segment regions of photoreceptor cells and it is this apical microvillous surface that is of particular interest with respect to phagocytosis. The present study is a report of a method to isolate a fraction that is enriched in microvilli from the apical surface of this highly polarized epithelial cell. Wheat germ agglutinin (WGA) conjugated sepharose beads are used to remove the microvillous membranes which are then observed with scanning and transmission electron microscopy. The proteins of this RPE-subfraction are separated through use of SDS-polyacrylamide gel electrophoresis. The relative molecular weights (Mr) and lectin binding properties of glycoproteins are examined in Western blots through the use of lectin-peroxidase conjugates as probes for carbohydrate residues. A preliminary comparison of membranes isolated from Long Evans (normal) and Royal College of Surgeons (dystrophic) rat retina RPE shows that the glycoproteins in these two preparations are different with respect to the binding of Concanavalin-A (Con-A) and WGA. In particular a glycoprotein in the normal RPE preparation with a Mr of 175K binds Con-A and WGA, but in the dystrophic RPE preparation binds little or no WGA. A glycoprotein present in the normal RPE preparation with a Mr of 86K binds Con-A and WGA, but both lectins have reduced binding sites in the dystrophic preparation. Limax flavus agglutinin (specific for sialic acid residues) binds to a high molecular weight glycoprotein with a Mr of 195K-196K which is present in both normal and dystrophic RPE membrane preparations and which also binds Con-A and WGA.

Examination of sialic acid binding on dystrophic and normal retinal pigment epithelium

In order to study differences in cell-surface sugars which may be involved in the phagocytic defect in Royal College of Surgeons (RCS) retinas, we have examined the presence or absence of lectin binding to carbohydrates on retinal pigment epithelial (RPE) plasma membranes of dystrophic (RCS-p+) and normal (Long-Evans) rats. A lectin which binds to both sialic acid and N-acetylglucosamine sugar residues, wheat germ agglutinin-ferritin (WGA-fe), was used. The specificity of WGA-fe binding to each sugar was studied by either pre-treating the tissue with neuraminidase enzyme which removes sialic acid residues, or by incubating the WGA-fe lectin with one of the haptens, N-acetylglucosamine. In non-enzyme-treated tissue, RPE cell-surface membranes from RCS retinas were densely labeled with WGA-fe as compared with the labeling on normal RPE, which appeared less dense and patchy in distribution. Wheat germ agglutinin-ferritin labeling in the presence of N-acetylglucosamine was blocked on both RCS and normal RPE surface membranes. After pre-treatment with neuraminidase, WGA-fe labeling on dystrophic RPE membranes was similar to non-enzyme-treated tissue but was enhanced on normal RPE. Labeling was blocked when N-acetylglucosamine was present with the lectin after enzyme pre-treatment. Other lectins which specifically bind to sialic acid, Limulus polyphemus agglutinin-ferritin (LPA-fe) and Limax flavus agglutinin (LFA) demonstrated sparse or no labeling on both RCS and normal RPE membranes. Our data suggests that N-acetylglucosamine residues predominate on RCS and normal RPE cell-surface membranes and that sialic acid binding sites are either not accessible to the lectins or may not be present.

Wheat germ agglutinin and concanavalin a binding during epithelial wound healing in the cornea

It has been hypothesized that there are differences between membrane-associated glycoconjugates of wounded (migrating) epithelium and those of nonwounded (stationary) epithelium. To test this hypothesis, wheat germ agglutinin-ferritin (WGA-Fe) and concanaval in A-ferritin (Con A-Fe) binding to apical membranes of wounded and nonwounded rabbit corneal epithelia were compared. Epithelial abrasions of the superior half of the cornea were allowed to heal in vivo for six hours. Fixed corneas were then incubated with lectin-ferritin and prepared for electron microscopy. Measurements (ferritin particles per linear um of membrane) of WGA-Fe indicated that binding to leading cells (40.7 particles/um), to areas 20 to 35 cells behind the leading edge (46.5 particles/um) and to nonwounded epithelium (45.1 particles/um) from contralateral eyes were not significantly different. A competitive inhibitor of WGA, 0.2M N-acetylglucosamine, however, blocked 94 percent of WGA binding on leading cells (2.3 particles/um), while binding persisted in areas behind the leading edge (39.5 particles/um) and on nonwounded epithelium (43.6 particles/um). This indicates that leading cell surfaces have a weak affinity for WGA. Unlike WGA, Con A showed a distinct preference for leading-edge cells (33.9 particles/um) compared to nonwounded epithelium (9 particles/um). In areas 20-35 cells behind the leading edge, Con A binding was intermediate to these two extremes.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative freeze-fracture and filipin-binding study of retinal pigment epithelial-cell basal membranes in diabetic rats

Breakdown of the blood-retinal barrier in diabetes may be related to alterations in the retinal pigment epithelial (RPE) cell layer. Morphological studies suggest increased permeability of diabetic RPE plasma membranes, and proliferation and flattening of the RPE basal infoldings have been observed in diabetic animals. In order to determine whether these phenomena are associated with changes in membrane protein or sterol composition, we have used quantitative electron-microscope freeze-fracture and filipin-binding techniques to study the RPE basal membrane in streptozotocin diabetic and 3-O-methyl glucose control rats. Perfusion-fixed retinas were processed for freeze-fracture and filipin-binding analysis. Filipin, a polyene antibiotic, binds specifically to 3-beta-hydroxy-sterols to produce membrane deformations recognizable by freeze-fracture. These analyses revealed an 11% increase in the density of intramembrane particles within the cytoplasmic (P-face) leaflet in diabetic rats as compared with the controls (P less than 0.01, t test). The increase occurred primarily in 6-9-nm particles, while smaller particles were decreased (P less than 0.001, chi-square test). Filipin binding was the same in both groups. These results suggest that alterations in intrinsic membrane proteins may contribute to permeability and surface area changes in the diabetic RPE but that RPE membrane sterols are not affected by diabetes.

Acid phosphatase localization in normal and dystrophic retinal pigment epithelium.

SummaryIn this study acid phosphatase (ACPase) was localized in the retinal pigment epithelium (RPE) of normal and Royal College of Surgeons (RCS) rats pink-eyed and pigmented with inherited retinal dystrophy to determine differences in staining during the post-engulfment stages of phagocytosis using two substrates, Na-β-glycerophosphate and cytidine-5′-monophosphate. Staining was similar using either substrate and in the normal RPE the Golgi system, lysosomes and phagosomes were ACPase-positive. In the dystrophic RPE, which has a diminished capacity to phagocytose photoreceptor rod outer segments, ACPase staining was localized on melanosomes in the pigmented dystrophic and on the apical microvillous membranes in the pink-eyed dystrophic, but was not localized on phagosomes in either the pink-eyed or pigmented dystrophic RPE. Since only a few phagosomes were seen at any given time in dystrophic RPE in vivo, a tissue expiant system was used to examine the number of latex beads phagocytosed by normal and RCS RPE, as well as the number of phagosomes containing both beads and ACPase activity in the normal and mutant RPE. Our findings indicate that in the dystrophic, fewer phagosomes are ACPase-positive than in the normal, and that some enzyme may be inappropriately shunted to either the apical microvilli or to melanosomes instead of to phagolysosomes.

Phagocytic interactions of sialated glycoprotein, sugar, and lectin coated beads with rat retinal pigment epithelium

Latex beads coated with mucin (a sialic acid containing glycoconjugate), N-acetylglucosamine (GlcNAc), or with the lectins, succinylated wheat germ agglutinin (sWGA) (binds to GlcNAc) or Limax flavus agglutinin (LFA) (binds to sialic acid residues) were used as phagocytic particles. Phagocytosis of these coated beads by retinal pigment epithelial (RPE) explants was determined by bead uptake in normal (Long Evans) and dystrophic (Royal College of Surgeons, RCS/p+) rat retinas. Electron microscopy showed that beads coated with mucin or LFA lectin were not phagocytized by either dystrophic or normal RPE. sWGA-coated beads were phagocytized by both dystrophic and normal RPE, while GlcNAc-coated beads were taken up by dystrophic RPE only. Specificity of uptake for sWGA and GlcNAc bead coatings was shown by the reduction in the number of beads phagocytized in the presence of the appropriate competing sugar or lectin. The lack of phagocytic uptake of beads coated with a sialated glycoprotein or a sialic acid binding lectin suggests that sialic acid residues are not recognized as particulate ligands in this phagocytic assay. The data which show the uptake of beads coated with sWGA (binds only GlcNAc) together with results which showed WGA (binds both GlcNAc and sialic acid)-coated beads were not taken up, further suggest that GlcNAc residues may be involved in bead phagocytosis. The most striking difference between normal and dystrophic RPE engulfment of coated beads is the uptake of GlcNAc-coated beads by dystrophic RPE only. These results suggest that the receptor molecules on dystrophic RPE cell surface membranes may be different from those on normal RPE membranes.