Genetic ablation of N‐linked glycosylation reveals two key folding pathways for R345W fibulin‐3, a secreted protein associated with retinal degeneration

ABSTRACT 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.—Hulleman, J. D., Kelly, J. W. Genetic ablation of N‐linked glycosylation reveals two key folding pathways for R345W fibulin‐3, a secreted protein associated with retinal degeneration. FASEB J. 29, 565‐575 (2015). www.fasebj.org