concanavalin-a and Retinal-Degeneration

An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.

Topics: Adenoviridae; Cell Line; Concanavalin A; Corneal Dystrophies, Hereditary; Cross-Linking Reagents; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Extracellular Matrix Proteins; Glycosylation; Homeostasis; Humans; Lectins; Macular Degeneration; Models, Genetic; Mutation; Optic Disk Drusen; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Tunicamycin

Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein roml. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin..rom1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.

Topics: Animals; Concanavalin A; Dimerization; Eye Proteins; Gene Expression; Glycosylation; Immunoblotting; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron; Nerve Tissue Proteins; Peripherins; Phenotype; Retinal Degeneration; Rod Cell Outer Segment; Sepharose; Tetraspanins; Transgenes

Topics: Animals; Concanavalin A; Electrophoresis; Glycoconjugates; Lectins; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Plant Lectins; Receptors, Mitogen; Reference Values; Retina; Retinal Degeneration; Wheat Germ Agglutinins

Topics: Animals; Cell Membrane; Concanavalin A; Disease Models, Animal; Ferritins; Methylmannosides; Photoreceptor Cells; Pigment Epithelium of Eye; Protein Binding; Rats; Rats, Inbred Strains; Retinal Degeneration; Retinitis Pigmentosa