Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with 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.
Departments of Chemistry and the Skaggs Institute for Chemical Biology andMolecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA john.hulleman@utsouthwestern.edu jkelly@scripps.edu.
John D
JD
Hulleman
Departments of Chemistry and the Skaggs Institute for Chemical Biology andMolecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA john.hulleman@utsouthwestern.edu jkelly@scripps.edu.
Jeffery W
JW
Kelly
D000256:Adenoviridae / Q000378:metabolism; D002460:Cell Line; D003208:Concanavalin A / Q000737:chemistry; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006706:Homeostasis; D006801:Humans; D037102:Lectins / Q000737:chemistry; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D015594:Optic Disk Drusen / Q000151:congenital; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012160:Retina / Q000378:metabolism; D012162:Retinal Degeneration / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000166:cytology; D014415:Tunicamycin / Q000737:chemistry
D003432:Cross-Linking Reagents; C119630:EFEMP1 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins; C000717247:HSPA5 protein, human; D037102:Lectins; D003208:Concanavalin A; D014415:Tunicamycin
EFEMP1; endoplasmic reticulum; malattia leventinese; unfolded protein response